Identification of SPRED2 as a Novel Regulator of Hypothalamic‐Pituitary‐Adrenal Axis Activity and of Body Homeostasis
Dissertation zur Erlangung des
naturwissenschaftlichen Doktorgrades
der Julius-Maximilians-Universität Würzburg
vorgelegt von
Melanie Ullrich
aus Karlstadt
Würzburg 2014
Eingereicht bei der Fakultät für Biologie am:
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Mitglieder der Promotionskommission:
Vorsitzender: ______
1. Gutachter: ______
2. Gutachter: ______
Tag des Promotionskolloquiums:
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Doktorurkunde ausgehändigt am:
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Eidesstattliche Erklärung
Hiermit erkläre ich an Eides statt, dass ich meine Dissertation mit dem Titel „Identification of SPRED2 as a Novel Regulator of Hypothalamic-Pituitary-Adrenal Axis Activity and of Body Homeostasis“ eigenständig, d.h. insbesondere selbstständig und ohne Hilfe einer kommerziellen Promotionsberatung angefertigt habe und keine anderen als die von mir angegebenen Quellen oder Hilfsmittel benutzt habe.
Des Weiteren versichere ich, dass ich die Gelegenheit zum Promotionsvorhaben nicht kommerziell vermittelt bekommen habe und dass ich insbesondere nicht eine Person oder Organisation eingeschaltet habe, die gegen Entgelt Betreuer bzw. Betreuerinnen für die Anfertigung von Dissertationen sucht.
Ich erkläre außerdem, dass die Regeln der Universität Würzburg über gute wissenschaftliche Praxis eingehalten wurden, dass die Dissertation weder in gleicher noch in ähnlicher Form bereits in einem anderen Prüfungsverfahren vorgelegen hat und dass ich akademische Grade weder bereits früher erworben noch zu erwerben versucht habe.
Würzburg, 2014 ______Melanie Ullrich
Die vorliegende Arbeit wurde unter Betreuung von Herrn Prof. Dr. Kai Schuh am Physiologischen Institut der Julius-Maximilians-Universität Würzburg angefertigt.
Teile dieser Dissertation wurden bereits in wissenschaftlichen Publikationen oder in Form von Abstracts zur Präsentation auf wissenschaftlichen Kongressen veröffentlicht.
Publikationen
Wissenschaftliche Publikationen Benz, P.M., Merkel, C.J., Offner, K., Abeßer, M., Ullrich, M., Fischer, T., Bayer, B., Wagner, H., Gambaryan, S., Ursitti, J.A., Adham, I.M., Linke, W., Feller, S.M., Fleming, I., Renné, T., Frantz, S., Unger, A., Schuh, K. (2013): Mena/VASP and αII-Spectrin complexes regulate cytoplasmic actin networks in cardiomyocytes and protect from conduction abnormalities and dilated cardiomyopathy. Cell Commun Signal. 11:56.
Ullrich, M., Bundschu, K., Benz, P.M., Abesser, M., Freudinger, R., Fischer, T., Ullrich, J., Renné, T., Walter, U., and Schuh, K. (2011): Identification of SPRED2 (Sprouty-related protein with EVH1 domain 2) as a negative regulator of the hypothalamic-pituitary-adrenal axis. J Biol Chem. 286 (11): 9477-88.
Ullrich, M. and Schuh, K. (2009): Gene trap - knockout on the fast lane. Methods Mol Biol. 561, 145-159.
Johne, J., Blume, C., Benz, P.M., Pozgajová, M., Ullrich, M., Schuh, K., Nieswandt, B., Walter, U., and Renné, T. (2006): Platelets promote coagulation factor XII-mediated proteolytic cascade systems in plasma. Biol Chem. 387, 173-78.
Bundschu, K., Knobeloch, K.-P., Ullrich, M., Schinke, T., Amling, M., Engelhardt, C.M., Renné T., Walter, U., and Schuh, K. (2005): Gene disruption of Spred-2 causes dwarfism. J Biol Chem. 280 (31), 28572-80.
Wissenschaftliche Publikationen in Revision Fischer, T., Rokita, A.G., Benz, P.M., Fissler, B., Ullrich, M., Abeßer, M., Seibold, S., Reichle, J., Lars, S., Maier, L.S., Ritter, O., Seidlmayer, L.K., Zuern, C., Wohlschlaeger, J., Baba, H.A., Schuh, K.: ATIP1 is a suppressor of cardiac hypertrophy and modulates AT2-dependent signaling in cardiac myocytes.
Andere wissenschaftliche Artikel in den Medien http://www.nationalgeographic.de/aktuelles/koerpereigene-stressbremse-entdeckt http://www.innovationsreport.de/html/berichte/biowissenschaften_chemie/stressbremse_versagt_172042.html http://derstandard.at/1297820667155/Forscher-identifizieren-Stressbremse
Abstracts zur Präsentation auf wissenschaftlichen Kongressen
Ullrich, M., Weber, M., Post, A., Popp, S., Guerrero, H., and Schuh. K. (2014): SPRED2 – a Critical Regulator of Synaptic Protein Expression and of Behavior? Posterpräsentation, Jahrestagung der Deutschen Physiologischen Gesellschaft, Mainz
Augustin, A.M., Ullrich M., Abesser, M., and Schuh K. (2014): Antagonizing aldosterone reduces cardiac effects of SPRED2-deficiency only partially. Posterpräsentation, Jahrestagung der Deutschen Physiologischen Gesellschaft, Mainz
Ullrich, M., Abesser, M., Augustin, A.M., and Schuh, K. (2013): SPRED2 - A Novel Modulator of Cardiac Hypertrophy and Electrical Conduction in the Heart? Posterpräsentation, Jahrestagung der Deutschen Physiologischen Gesellschaft, Heidelberg
Ullrich, M., Abesser, M., Augustin, A.M., and Schuh, K. (2012): SPRED2 - A Novel Modulator of Cardiac Hypertrophy and Electrical Conduction in the Heart? Posterpräsentation, Jahrestagung des Deutschen Zentrums für Herzinsuffizienz, Kloster Banz
Ullrich, M. and Schuh, K. (2012): Physiological Role of SPRED2 in the Mammalian Heart. Vortrag, Begutachtung des DZHI-Projekts B10, ESAB-Meeting des DZHI, Würzburg
Ullrich, M. and Schuh, K. (2011): Identification of SPRED2 as a negative regulator of the HPA axis. Posterpräsentation, Jahrestagung der Deutschen Physiologischen Gesellschaft, Regensburg
Bundschu, K., Schuh, K., Walter, U., and Ullrich, M. (2010): SPRED2 (Sprouty-related protein with EVH1 domain 2) regulates bone growth and hypothalamic-pituitary-adrenal hormone secretion. Posterpräsentation, EMBO Meeting, Barcelona
Ullrich, M., Bundschu, K., Walter, U., and Schuh, K. (2010): Dysregulated hypothalamic- pituitary-adrenal (HPA) hormone secretion in SPRED2-deficient mice. Posterpräsentation, Jahrestagung der Deutschen Physiologischen Gesellschaft, Kopenhagen.
Ullrich, M., Walter, U., and Schuh, K. (2009): Dysregulated hypothalamic-pituitary-adrenal (HPA) hormone secretion in SPRED2-deficient mice. Posterpräsentation, Jahrestagung der Deutschen Vereinten Gesellschaft für Klinische Chemie und Laboratoriumsmedizin, Leipzig
Ullrich, M., Bundschu, K., Freudinger, R., Gattenloehner, S., Walter, U., and Schuh, K. (2009): Dysregulation of hypothalamic-pituitary-adrenal hormone secretion in SPRED2-deficient mice. Posterpräsentation, Jahrestagung der Deutschen Physiologischen Gesellschaft, Giessen.
Ullrich, M., Freudinger, R. Bundschu, K., Walter, U., and Schuh, K. (2008): Phenotype puzzle in Spred-2-deficient mice. Posterpräsentation, Jahrestagung der Deutschen Vereinten Gesellschaft für Klinische Chemie und Laboratoriumsmedizin, Mannheim
Ullrich, M., Bundschu, K., Freudinger, R., Gattenloehner, S., Walter, U., and Schuh, K. (2008): Hormone, ion, and fluid imbalance in Spred-2-/- mice. Posterpräsentation, Graduate School of Life Sciences, Würzburg
Ullrich, M., Bundschu, K., Walter, U., and Schuh, K. (2008): Phenotype puzzle in Spred-2- deficient mice. Posterpräsentation, Jahrestagung der Deutschen Physiologischen Gesellschaft, Köln.
Ullrich, M., Bundschu, K., Gattenloehner, S., Walter, U., and Schuh, K. (2007): Dwarfism and kidney failure in Spred-2-deficient mice. Posterpräsentation, Jahrestagung der Deutschen Physiologischen Gesellschaft, Hannover.
Ullrich, M., Bundschu, K., Walter, U., and Schuh, K. (2006): Spred-2 deficiency results in dwarfism and kidney failure. Vortrag und Posterpräsentation, Experimental Biology Meeting, San Francisco
Ullrich, M., Schmitteckert, E., Renné, T., Walter, U., and Schuh, K. (2006): Generation of a system for thrombocyte-specific gene deletion. Posterpräsentation, Jahrestagung der Gesellschaft für Thrombose und Hämostase, Basel
Preise und Auszeichnungen
„1. Posterpreis“ auf der Jahrestagung des Deutschen Zentrums für Herzinsuffizienz (DZHI) in Kloster Banz
„FEPS Best Poster Award” auf dem European Young Physiologists Symposium im Rahmen der Physiologentagung in Kopenhagen
„Travel Award” im Rahmen des Experimental Biology Meetings in San Francisco
Das Genie beginnt die schönen Werke, aber nur die Arbeit vollendet sie.
Joseph Joubert
Meiner Familie
Danksagung
An dieser Stelle möchte ich mich bei all den Personen bedanken, ohne die die Durchführung und Vollendung meiner Dissertation nicht möglich gewesen wäre:
Mein größter Dank gilt Herrn Prof. Dr. Kai Schuh, der mir die Bearbeitung dieses Dissertationsthemas in seiner Arbeitsgruppe ermöglicht hat. Lieber Kai, du warst mir in all den Jahren ein ganz hervorragender Betreuer. Ich konnte sowohl in wissenschaftlicher als auch in menschlicher Hinsicht wirklich sehr viel von dir lernen. Ganz herzlichen Dank für die Freiheit sowohl bei der experimentellen als auch bei der schriftlichen Gestaltung dieser Arbeit, für deine stets offene Tür, für deine unerschöpfliche Diskussionsbereitschaft, für deine Geduld, deine Motivation und deinen Glauben an mich. Durch deine immerwährende Unterstützung konnte ich die Ergebnisse dieses schönen Projekts publizieren und als meine Doktorarbeit zu Papier bringen.
Ein herzliches Dankeschön gilt auch Herrn Prof. Dr. Georg Krohne für die Bereitschaft, nach meiner Diplomarbeit auch meine Dissertation als Vertreter der biologischen Fakultät zu begutachten. Vielen Dank für die stets unkomplizierte Zusammenarbeit.
Ganz besonders bedanken möchte ich mich bei allen aktuellen und ehemaligen Kolleginnen und Kollegen der AG Schuh. Lieben Dank an euch, Doreen, Krissi, Anne, Ruth, Priscilla, Carla, Feli, Chris, Constanze, Marco, Peter, Tobi, Hans und Robert für das stets konstruktive und freundschaftliche Miteinander im Labor. Auch die vielen schönen Unternehmungen zusammen mit euch außerhalb des Labors, wie die Cocktailabende im Joes, die diversen (Geburtstags-) Partys, der Besuch der Wilhelma in Stuttgart mit gemütlichem Ausklang im Bierzelt auf dem Cannstatter Wasen und die Bootstour auf dem Main werden mir immer in Erinnerung bleiben. Vielen Dank an die AG von Prof. Dr. Michaela Kuhn mit Birgit, Katharina, Sabrina, Heike, Franziska, Annett, Thomas, Hitoshi und Wen. Danke euch allen für das fröhliche Arbeitsklima und für die unterhaltsamen Mittagspausen. Ganz lieben Dank auch an die AG von Prof. Dr. Andreas Friebe mit Barbara, Dieter, Ronald und Noomen. Speziell dir, liebe Barbara, möchte ich ganz herzlich für die seelische Unterstützung und die vielen lieben und aufmunternden Worte besonders gegen Ende meiner Promotionsphase danken. Unvergesslich werden für mich die abenteuerlichen Bierwanderungen, die legendären Weihnachtsfeiern mit Schrottwichteln, die lustigen Skiausflüge nach Fügen und nach Au und natürlich auch die geselligen Sommerabende auf den Weinfesten Würzburgs mit euch bleiben. Weiterhin danken möchte ich den Tierpflegerinnen Iris, Kerstin und Evi für die unkomplizierte und freundschaftliche Zusammenarbeit und für die gute und umfassende Betreuung meiner nicht ganz so pflegeleichten SPRED2 KO-Mauslinie.
Ganz kuscheligen Dank an Katze Floh und Kater Kalle, für die seelische Unterstützung beim Zusammenschreiben dieser Arbeit in Form von beruhigendem Schnurren auf meinem Schoß oder auf dem Schreibtisch. Ich danke euch auch für das ausgiebige Schlafen auf der Computertastatur, die so immer warm gehalten wurde, auch wenn es mit dem Schreiben mal nicht so lief.
Zu guter Letzt möchte ich von ganzem Herzen meiner Familie danken. Liebe Julia, danke für die Überraschungspäckchen per Hauspost und für die vielen Telefonate in guten und in schlechten Zeiten. Ich denke, das hat bei uns beiden einiges bewegt…und ich zieh es jetzt durch! Liebe Mama, lieber Papa, herzlichen Dank, dass ihr immer bei mir im Labor vorbeigeschaut habt, wenn ihr in Würzburg wart, für den alljährlichen Besuch auf dem Würzburger Weihnachtsmarkt mit Oma und für das viele gute Essen, das ihr jedesmal für mich mitgebracht habt. Danke für eure immer dagewesene Unterstützung, für das Vertrauen und den Rückhalt, den ihr mir gebt und gegeben habt, diese akademische Karriere zu beginnen und bis zum Doktortitel fortzuführen.
Table of Contents
Summary 1 ...... 1
Zusammenfassung 2 ...... 3
Introduction 3 ...... 5
3.1. MAPK Signaling – Control of Proliferation and Growth ...... 5 3.1.1. The Ras/ERK/MAPK Pathway ...... 5 3.1.2. Ras/ERK/MAPK Pathway Dysregulation – Causes of Cancer and Rasopathies ...... 8 3.2. SPREDs – Inhibitors of the Ras/ERK/MAPK Pathway ...... 9 3.2.1. SPRED Isoforms ...... 9 3.2.2. SPRED Domain Structure and Functions ...... 10 3.2.2.1 The EVH1 Domain ...... 10 3.2.2.2 The c‐Kit Binding Domain ...... 11 3.2.2.3. The Sprouty Domain ...... 11 3.2.3. SPRED Expression Patterns and Localizations ...... 12 3.2.4. SPREDs and Suppression of Ras/ERK/MAPK Signaling ...... 14 3.2.5. Regulation of SPRED‐mediated Ras/ERK/MAPK Pathway Inhibition ...... 15 3.2.6. SPRED Functions in Mice and Men ...... 17 3.2.6.1. In vitro Studies and Mouse Models ...... 17 3.2.6.2. First Lessons from Humans ...... 22 3.3. The Mouse ‐ A Research Model for Human Gene Function and Disease ...... 24 3.3.1. Mutagenesis Strategies in Mice ...... 25 3.3.2. Gene Trapping ‐ Knockout on the Fast Lane ...... 27 3.4. Hormonal Regulation of Body Homeostasis ...... 29 3.4.1. The Hypothalamic‐Pituitary‐Adrenal Axis ...... 29 3.4.1.1. The Hypothalamus – Neural Stimuli Trigger the Release of CRH and AVP ...... 30 3.4.1.2. The Pituitary – CRH/AVP‐mediated ACTH Release from Anterior Lobe and Osmolality/Volume‐mediated AVP Release from Posterior Lobe ...... 32 3.4.1.3. The Adrenal – ACTH Regulates the Release of Glucocorticoids and Mineralocorticoids ...... 34 3.4.1.4. Feedback Regulation and Circadian Rhythm of the HPA Axis ...... 37 3.4.2. The Renin‐Angiotensin‐Aldosterone Axis ...... 38 3.4.2.1. The Renin‐Angiotensin System (RAS) ...... 38 3.4.2.2. Aldosterone ...... 40 3.4.2.3. RAS Feedback Regulation, Dysregulation, Antagonists, and Associated Diseases ...... 42 3.5. Aim of Thesis ...... 43
Materials and Methods 4 ...... 47
4.1. Mouse Handling ...... 47 4.1.1. Generation of SPRED2 KO Mice ...... 47 4.1.2. Mouse Raising and Breeding ...... 47 4.1.3. Genotyping of SPRED2‐deficient Mice ...... 48 4.2. Mouse Experiments ...... 49 4.2.1. Examination of Water Uptake ...... 50 4.2.2. Experimental Water Deprivation ...... 50 4.2.3. Blood Collection and Serum Preparation ...... 50 4.2.4. Determination of Routine Laboratory Serum Parameters ...... 51 4.2.5. Determination of Hormone Levels ...... 51 4.2.6. Wound Control and Monitoring ...... 52 4.3. Cloning of cDNAs and Expression Plasmids ...... 52 4.3.1. pcDNA3.1‐EVH1‐β‐geo and pcDNA3.1‐β‐geo ...... 52
4.3.2. pGL3‐CRHProm‐RE and pGL3‐CRHProm ...... 54 4.4. Cell Culture, Transfection, and in vitro Assays ...... 55 4.4.1. Cultivation of HEK 293 T cells ...... 55 4.4.2. Cultivation of mHypoE‐44 cells...... 56 4.4.3. Transfection of HEK 293 T and mHypoE‐44 cells ...... 56 4.4.4. In vitro ERK Activity Assay ...... 57 4.4.5. In vitro CRH Promoter Reporter Assay ...... 57 4.4.6. In vitro Ets Transcription Factor Reporter Assay ...... 58 4.5. Analysis and Quantification of Protein and mRNA Expression ...... 58 4.5.1. Western Blot ...... 58 4.5.2. Northern Blot ...... 60 4.5.3. Quantification of ERK Phosphorylation and of CRH mRNA Levels in Brain ...... 61 4.6. Histology and Immunohistochemistry ...... 62 4.6.1. Hematoxylin and Eosin Staining (H&E Staining) ...... 62 4.6.2. TUNEL Assay ...... 62 4.6.3. Immunohistochemistry ...... 63 4.6.4. X‐Gal Staining ...... 63 4.7. Statistics ...... 64 4.7.1. Basic Statistics ...... 64 4.7.2. Kaplan‐Meier Survival Analysis ...... 64
Results 5 ...... 67
5.1. Generation of SPRED2 KO Mice ...... 67 5.2. Breeding and Genotyping of SPRED2 KO Mice ...... 69 5.3. The EVH1‐β‐geo Fusion Protein Expressed in SPRED2 KO Mice May Act Dominant Negative ...... 70 5.3.1. Deletion of SPRED2 but Expression of an EVH1‐β‐geo Fusion Protein ...... 71 5.3.2. Functional Expression of Subcloned EVH1‐β‐geo in Eukaryotic Cells ...... 72 5.3.3. Putative Dominant Negative Effect of EVH1‐β‐geo on SPRED1 and Ras/ERK/MAPK Signaling in SPRED2 KO Mice ...... 74 5.4. Obvious and Versatile Phenotype in SPRED2 KO Mice ...... 75 5.5. SPRED2 Deficiency in Mice Leads to Hydronephrosis, Renal Atrophy, and Kidney Deterioration .. 75 5.6. Hydronephrosis Is Associated with Tubular Breakdown and Apoptosis in SPRED2 KO Mice ...... 77 5.7. Kidney Damage in SPRED2 KOs Is Accompanied by Polydipsia, Hyperosmolality, and Higher Serum Salt Load ...... 79 5.8. Serum Parameter Analyses Underline Kidney Damage and Indicate SPRED2 Deficiency‐Provoked Homeostatic Imbalance ...... 80 5.9. Loss of SPRED2 Leads to Dysregulation of Salt and Water Homeostasis‐Controlling Hormones .... 83 5.9.1. Augmented Aldosterone Secretion Is Accompanied by Elevated Expression of Aldosterone‐ Synthase ...... 83 5.9.2. Hyperaldosteronism Develops Independently of Renin‐Angiotensin System Activity ...... 84 5.9.3. Water Deprivation Causes Elevated Loss of Body Fluid in SPRED2 KO Mice ...... 85 5.9.4. Vasopressin Response Is Unaffected by Ablation of SPRED2 ...... 88 5.10. Increased Release of the Stress Hormones CRH, ACTH, and Corticosterone from the Hypothalamic‐Pituitary‐Adrenal Axis in SPRED2 KO Mice ...... 89 5.11. Augmented Hypothalamic‐Pituitary‐Adrenal Stress Hormone Release Is Accompanied by Elevated Hypothalamic‐Pituitary Growth Hormone Secretion in SPRED2 KO Mice ...... 90 5.12. Overshooting Stress Hormone Release Is Associated with Obsessive Grooming and Self‐Inflicted Skin Lesions in SPRED2 KO Mice ...... 91 5.13. SPRED2 Expression Profiling in Hypothalamic‐Pituitary‐Adrenal Tissues and Kidney ...... 93 5.14. SPRED2 Inhibits Hypothalamic CRH Production and Release by Downregulating CRH Promoter Activity in an ERK/MAPK/Ets1‐Dependent Manner ...... 95 5.14.1. Increased in vivo ERK Phosphorylation in Brains of SPRED2 KOs ...... 95 5.14.2. SPREDs Suppress CRH Transcription in vitro ...... 96 5.14.3. SPREDs Inhibitory Effect on CRH Promoter Activity is Dependent on ERK/MAPK‐mediated Regulation of Ets Transcription Factors ...... 98 5.14.4. SPREDs Reduced CRH Release from mHypoE‐44 Cells in vitro ...... 99 5.14.5. Higher Content of CRH mRNA in Hypothalamic PVN‐Containing Brain Regions of SPRED2‐deficient Mice ...... 100 5.15. Reduced Survival Probability in SPRED2 KO Mice ...... 101
Discussion 6 ...... 103
6.1. The SPRED2 Gene Trap Model – Possible Effects of the EVH1‐β‐geo Fusion Protein ...... 103 6.2. Causes of Kidney Damage in SPRED2 KO Mice – An Interplay of Hydronephrosis, Atrophy, and Apoptosis ...... 106 6.3. Polydipsia, Impaired Water Retention, and Hyperosmolality in SPRED2 KOs ‐ No Effect of SPRED2 on AVP‐Dependent Water Balance Regulation ...... 108 6.4. Hyperaldosteronsim – The Elicitor of Hyperosmolality and Salt and Water Imbalances in SPRED2 KO Mice ...... 109 6.5. Increased Aldosterone‐Synthase Expression and Elevated Serum ACTH – Causes of Renin‐ Angiotensin System‐Independent Hyperaldosteronism ...... 110 6.6. Hyperactivity of the HPA Axis in SPRED2‐deficient Mice – Elevated Release of Stress and Growth Hormones ...... 113 6.7. Excessive Grooming in SPRED2 KOs ‐ A Result of HPA Axis Overactivity and Evidence of Endogenous Stress ...... 115 6.8. SPRED2 ‐ A Novel Regulator of CRH Gene Transcription and Release ...... 116 6.9. SPRED1 and SPRED2 Deficiency ‐ Redundancy in Protein Function and Impact on Cancer and Rasopathies ...... 118 Conclusion and Outlook 7 ...... 123
7.1. SPRED2 – A Critical Mediator of Synaptic Transmission and Regulator of Behavior? ...... 125 7.2. SPRED2 – A Novel Regulator of Cardiac Hypertrophy and Electrical Conduction in the Heart? .... 126 7.3. SPRED2 Deficiency – A Cause of Rasopathy in Humans? ...... 128
References 8 ...... 131
Appendix 9 ...... 157
9. 1. Genotyping ...... 157 9.2. EVH1‐β‐geo Fusion Protein ...... 165 9.3. CRH Promoter Reporter Assay ...... 168 9.4. Vector Cards ...... 173 9.5. 4xEts1 Reporter Assay ...... 178 9.6. Transcription Factor Binding Site Prediction in the Murine CRH Promoter ...... 181 9.7. Northern Blot ...... 195 9.8. Abbreviations ...... 197
Summary 1
SPRED proteins are inhibitors of the Ras/ERK/MAPK signaling pathway, an evolutionary highly conserved and very widespread signaling cascade regulating cell proliferation, differentiation, and growth. To elucidate physiological consequences of SPRED2 deficiency, SPRED2 KO mice were generated by a gene trap approach. An initial phenotypical characterization of KO mice aged up to five months identified SPRED2 as a regulator of chondrocyte differentiation and bone growth. Here, the loss of SPRED2 leads to an augmented FGFR-dependent ERK activity, which in turn causes hypochondroplasia-like dwarfism. However, long term observations of older KO mice revealed a generally bad state of health and manifold further symptoms, including excessive grooming associated with severe self-inflicted wounds, an abnormally high water uptake, clear morphological signs of kidney deterioration, and a reduced survival due to sudden death. Based on these observations, the aim of this study was to discover an elicitor of this complex and versatile phenotype. The observed kidney degeneration in our SPRED2 KO mice was ascribed to hydronephrosis characterized by severe kidney atrophy and apoptosis of renal tubular cells. Kidney damage prompted us to analyze drinking behavior and routine serum parameters. Despite polydipsia, which was characterized by a nearly doubled daily water uptake, the significantly elevated Na+ and Cl- levels and the resulting serum hyperosmolality could not be compensated in SPRED2 KOs. Since salt and water balance is primarily under hormonal control of aldosterone and AVP, we analyzed both hormone levels. While serum AVP was similar in WTs and KOs, even after experimental water deprivation and an extreme loss of body fluid, serum aldosterone was doubled in SPRED2 KO mice. Systematic investigation of contributing upstream hormone axes demonstrated that hyperaldosteronism developed independently of an overactivated Renin- Angiotensin system as indicated by halved serum Ang II levels in KO mice. However, aldosterone synthase expression in the adrenal gland was substantially augmented. Serum corticosterone, which is like aldosterone released from the adrenal cortex, was more than doubled in SPRED2 KOs, too. Similar to corticosterone, the production of aldosterone is at least in part under control of pituitary ACTH, which is further regulated by upstream hypothalamic
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1. Summary
CRH release. In fact, stress hormone secretion from this complete hypothalamic-pituitary- adrenal axis was upregulated because serum ACTH, the mid acting pituitary hormone, and hypothalamic CRH, the upstream hormonal inductor of HPA axis activity, were also elevated by 30% in SPRED2 KO mice. This was accompanied by an upregulated ERK activity in paraventricular nucleus-containing hypothalamic brain regions and by augmented hypothalamic CRH mRNA levels in our SPRED2 KO mice. In vitro studies using the hypothalamic cell line mHypoE-44 further demonstrated that both SPRED1 and SPRED2 were able to downregulate CRH promoter activity, CRH secretion, and Ets factor-dependent CRH transcription. This was in line with the presence of various Ets factor binding sites in the CRH promoter region, especially for Ets1. Thus, this study shows for the first time that SPRED2-dependent inhibition of Ras/ERK/MAPK signaling by suppression of ERK activity leads to a downregulation of Ets1 factor-dependent transcription, which further results in inhibition of CRH promoter activity, CRH transcription, and CRH release from the hypothalamus. The consecutive hyperactivity of the complete HPA axis in our SPRED2 KO mice reflects an elevated endogenous stress response becoming manifest by excessive grooming behavior and self-inflicted skin lesions on the one hand; on the other hand, in combination with elevated aldosterone synthase expression, this upregulated HPA hormone release explains hyperaldosteronism and the associated salt and water imbalances. Both hyperaldosteronism and polydipsia very likely contribute further to the observed kidney damage. Taken together, this study initially demonstrates that SPRED2 is essential for the appropriate regulation of HPA axis activity and of body homeostasis. To further enlighten and compare consequences of SPRED2 deficiency in mice and particularly in humans, two follow-up studies investigating SPRED2 function especially in heart and brain, and a genetic screen to identify human SPRED2 loss-of-function mutations are already in progress.
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Zusammenfassung 2
SPRED-Proteine sind Inhibitoren des hochkonservierten und in allen Geweben verbreiteten Ras/ERK/MAPK-Signalwegs, welcher Proliferation, Differenzierung und das Wachstum von Zellen reguliert. Um physiologische Konsequenzen der SPRED2-Defizienz im lebenden Modellorganismus aufzuklären, haben wir SPRED2-KO-Mäuse mithilfe der „gene trap“-Methode generiert. Eine erste Studie zur phänotypischen Charakterisierung mit KO-Mäusen bis zu einem Alter von fünf Monaten identifizierte SPRED2 als Regulator der Chondrozytendifferenzierung und des Knochenwachstums. So bewirkt der Verlust der SPRED2-Proteinfunktion eine erhöhte FGFR-vermittelte ERK-Aktivität, was wiederum einen Hypochondroplasie-ähnlichen Minderwuchs verursacht. Allerdings offenbarten Langzeitbeobachtungen älterer KO-Mäuse einen im Allgemeinen sehr schlechten Gesundheitszustand und weitere facettenreiche Symptome, darunter exzessives Putzverhalten mit schweren, selbst zugefügten Wunden, einen abnorm hohen täglichen Wasserkonsum, klare morphologische Anzeichen einer Nierenschädigung und eine reduzierte Überlebenswahrscheinlichkeit durch plötzlichen Tod. Ziel dieser Studie war es, basierend auf unseren Beobachtungen, einen Auslöser für diesen komplexen und vielseitigen Phänotyp zu finden. Die beobachtete Nierendegeneration in unseren SPRED2-KO-Mäusen war auf eine Hydronephrose zurückzuführen, welche durch schwere Atrophie des Nierengewebes und Apoptose von Nierentubuluszellen gekennzeichnet war. Aufgrund des Nierenschadens haben wir Trinkverhalten und gängige Serumparameter analysiert. Trotz der Polydipsie, die sich durch eine nahezu verdoppelte tägliche Wasseraufnahme manifestierte, konnten signifikant erhöhte Na+- und Cl--Werte und die daraus resultierende Hyperosmolalität im Serum der SPRED2-KOs nicht kompensiert werden. Weil Salz- und Wasserhaushalt zum größten Teil unter der hormonellen Kontrolle von Aldosteron und ADH stehen, haben wir beide Hormonspiegel untersucht. Während die ADH-Werte im Serum von WT- und KO-Mäusen vergleichbar waren, insbesondere nach experimentellem Wasserentzug und einem extremen Verlust von Körperflüssigkeit, waren die Serumspiegel von Aldosteron in den SPRED2-KO-Mäusen verdoppelt. Die systematische Untersuchung übergeordneter regulatorischer Hormonachsen 3
2. Zusammenfassung ergab, dass sich der Hyperaldosteronismus unabhängig von einer erhöhten Aktivität des Renin- Angiotensin-Systems entwickelte, da die Serum-Ang II-Spiegel in den SPRED2-KOs etwa um die Hälfte reduziert waren. Die Expression der Aldosteronsynthase in der Nebenniere war jedoch wesentlich erhöht. Für Kortikosteron, das wie Aldosteron von der Nebennierenrinde freigesetzt wird, konnten wir ebenfalls mehr als doppelt so hohe Werte im Serum der KO-Tiere detektieren. Die Aldosteron-Produktion steht, ähnlich wie bei Kortikosteron, zumindest teilweise unter der Kontrolle des hypophysären Hormons ACTH, dessen Sekretion wiederum übergeordnet durch die Freisetzung von CRH aus dem Hypothalamus geregelt wird. Tatsächlich war die Stresshormon-Sekretion entlang dieser gesamten Hypothalamus- Hypophysen-Nebennierenrinden-Achse erhöht, da Serum-ACTH, das mittlere, hypophysäre Hormon, und hypothalamisches CRH, der übergeordnete hormonelle Induktor der HPA-Achse, in den SPRED2-KOs auch um 30% erhöht waren. Zusätzlich waren die ERK-Aktivität ebenso wie die CRH-mRNA-Spiegel im paraventrikulären Nukleus des Hypothalamus in unseren SPRED2-KO-Mäusen deutlich höher. In vitro Studien mit der Hypothalamus-Zelllinie mHypoE- 44 zeigten weiterhin, dass sowohl SPRED1 als auch SPRED2 die Aktivität des CRH-Promotors, die CRH-Sekretion und die Ets-Faktor-abhängige CRH-Transkription reduzieren können. Passend dazu enthält die CRH-Promotorregion zahlreiche verschiedene Bindungsstellen für Transkriptionsfaktoren der Ets-Familie, speziell für Ets1. Somit zeigt diese Studie zum ersten Mal, dass die durch SPRED2-vermittelte Hemmung der Ras/ERK/MAPK-Signalkaskade mittels Unterdrückung der ERK-Aktivität zu einer Herunterregulation der Ets1-Faktor-abhängigen Transkription führt, was eine Hemmung der CRH-Promotoraktivität, der CRH-Transkription und der CRH-Freisetzung aus dem Hypothalamus zur Folge hat. Die daraus resultierende Hyperaktivität der gesamten HPA-Achse in unseren SPRED2-KO-Mäusen spiegelt eine erhöhte endogene Stress-Reaktion wider und äußert sich durch übermäßiges Putzverhalten und durch selbst zugefügte Hautläsionen auf der einen Seite; auf der anderen Seite erklärt dies, in Kombination mit der erhöhten Aldosteronsynthase-Expression, den Hyperaldosteronismus und das damit verbundene Ungleichgewicht in Salz- und Wasserhaushalt. Weiterhin tragen sowohl Hyperaldosteronismus als auch Polydipsie sehr wahrscheinlich zu den beobachteten Nierenschädigungen bei. Zusammengefasst ist diese Studie ein erster Hinweis, dass SPRED2 wesentlich an der adäquaten Regulation der HPA-Achsen-Aktivität beteiligt ist und essentiell ist für die Aufrechterhaltung der Homöostase im Körper. Um die Folgen von SPRED2-Defizienz in Mäusen und vor allem im Menschen weiter aufzuklären und zu vergleichen, erforschen wir in zwei Folgeprojekten die Funktion von SPRED2 speziell im Gehirn und im Herzen und führen parallel ein genetisches Screening zur Identifikation von funktionellen SPRED2-Mutationen im Menschen durch.
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Introduction 3
3.1. MAPK Signaling – Control of Proliferation and Growth
The mitogen-activated protein kinase (MAPK) cascades are evolutionary conserved pathways that respond to various extracellular signals in transmitting them to their intracellular targets. Within these cascades, the signal is propagated by phosphorylation and activation of sequential kinases, resulting in phosphorylation and activation of regulatory target proteins in cytoplasm and nucleus. Thereby, they control a large number of fundamental cellular processes including proliferation, differentiation, growth, motility, stress response, survival, and apoptosis (for review see (Plotnikov et al., 2011; Raman et al., 2007)). To date, four different mammalian MAPK cascades have been identified and named according to their corresponding MAPKs: extracellular signal-regulated kinase 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK), p38, and ERK5. Other kinases like ERK3/4 and ERK7/8 indeed share sequence similarities with the typical MAPKs but show a different mode of activation, suggesting that they are not components of genuine MAPK cascades (Coulombe and Meloche, 2007).
3.1.1. The Ras/ERK/MAPK Pathway
The MAPK pathway first elucidated and until today considered as the “classical” MAPK cascade is the ERK1/2 pathway (Figure 1). In contrast to other MAPK pathways, its function mainly concentrates on the regulation of cell proliferation and differentiation (Seger and Krebs, 1995). It is preferentially activated in response to mitogens and growth factors (GFs), e.g. epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), nerve growth factor (NGF), but also by cytokines like stem cell factor (SCF), interleukins (IL-3, IL5), and tumor necrosis factor α (TNF α). These various ligands bind to receptor tyrosine kinase (RTK) monomers, induce receptor dimerization
5
3. Introduction through monomer crosslinking and thereby the activation of the RTK. RTK monomers consist of an extracellular N-terminal region, a single hydrophobic transmembrane-spanning domain, and an intracellular C-terminal region (Ullrich and Schlessinger, 1990). The extracellular N-terminal region exhibits a variety of conserved elements, which are characteristic for each subfamily and of which the most occurring are immunoglobulin (Ig)-like domains and cysteine-rich regions (Ullrich and Schlessinger, 1990; van der Geer et al., 1994). It primarily contains the ligand binding site, and after extracellular ligand binding RTK activation is translated across the membrane into activation of intracellular domains. The intracellular C-terminal region comprises catalytic domains responsible for the kinase activity and thus catalyzes reciprocal autophosphorylation of RTK monomers on key tyrosine residues (Ullrich and Schlessinger, 1990). Via its Src (Sarcoma) homology 2 (SH2) domain, the adapter protein growth factor receptor-bound protein 2 (GRB2) binds to phosphotyrosine interaction modules of activated receptors (Lowenstein et al., 1992). Beyond direct interaction with tyrosine phosphorylated RTKs, GRB2 can also bind to intermediary adapter proteins, such as SHC (Src homology and collagen protein) (Skolnik et al., 1993), FRS2 (fibroblast growth factor receptor substrate 2) (Kouhara et al., 1997), IRS-1/2 (insulin receptor substrate 1/2) (Skolnik et al., 1993), GAB1 (GRB2-associated binding protein 1) (Holgado-Madruga et al., 1996), and SHP2 (Src homology 2 domain containing protein-tyrosine phosphatase 2, also PTPN11=Tyrosine-protein phosphatase non-receptor type 11) (Hadari et al., 1998). These adapters are associated with the receptor via SH2 or phosphotyrosine-binding (PTB) domains and are tyrosine phosphorylated upon binding.
Figure 1: The Ras/ERK/MAPK Pathway - A Critical Regulator of Cell Proliferation, Differentiation and Growth Stimulation of RTKs by GFs or cytokines results in the activation of the “classical” MAPKs ERK1 and ERK2. ERK further phosphorylates cytoplasmic and nuclear targets, which are able to regulate the transcription of target genes (CP = cytoplasmic proteins). Figure based on (Bundschu, 2005).
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3. Introduction
Via its Src homology 3 (SH3) domain, GRB2 also interacts with the proline-rich motif of SOS (son of sevenless) (Chardin et al., 1993). When the GRB2-SOS complex docks to tyrosine phosphorylated RTKs or adapter proteins at the cell membrane, SOS becomes activated. There, GRB2-SOS interacts with and activates Ras (rat sarcoma) (Egan et al., 1993). Ras comprises a family of small GTPases, which were identified as proto-oncogenes and of which H-Ras, N-Ras, and K-Ras are the most common family members (Malumbres and Barbacid, 2003). Ras contains an N-terminal G domain, which binds guanosine nucleotides, and a C- terminal CAAX box, which is lipid-modified and required for its plasma membrane targeting (Hancock et al., 1989; Temeles et al., 1985; Willumsen et al., 1984). SOS is a RasGEF (guanine nucleotide exchange factor) that activates Ras by catalyzing the exchange of bound GDP to GTP (Chardin et al., 1993). The activated GRB2-SOS complex binds exclusively to the inactive Ras-GDP-bound form, destabilizes the Ras-GDP binding, and promotes the dissociation of GDP from Ras. Because the cytosolic ratio of GTP is much higher than GDP (10:1), GTP associates with the GTPase, which results in the release of the GRB2-SOS complex and to the activation of Ras at the plasma membrane (Bos et al., 2007). Ras activates Raf (rapidly accelerated fibrosarcoma), the MAPKKK of the ERK pathway, which phosphorylates target kinases on serine and threonine kinase. The Raf family comprises three members, namely A-Raf, B-Raf, and C-Raf/Raf-1. Like Ras, Raf was identified as proto- oncogenic, whereas only B-Raf shows a high mutation rate in human cancers (Davies et al., 2002; Wellbrock et al., 2004). Raf kinases share a conserved domain structure containing an N- terminal regulatory domain and a C-terminal kinase domain (Wellbrock et al., 2004). Active Ras binds directly to the regulatory domain of Raf (Zhang et al., 1993) and thereby recruits the kinase to the plasma membrane, which is indispensable for its activation (Leevers et al., 1994; Stokoe et al., 1994). The phosphorylation and activation of Raf is Ras-independent and mediated by Src family protein-tyrosine kinases and other protein-serine/threonine kinases (King et al., 2001; Marais et al., 1995). Activated Raf catalyzes the activation of the MAPKKs MEK1 and MEK2 by phosphorylation on 2 serine residues (Kyriakis et al., 1992; Macdonald et al., 1993). MEK1/2 are dual specificity kinases that can act as both tyrosine and serine/threonine kinases. They are highly specific for their downstream components, the MAPKs ERK1 (also known as MAPK3 or p44 MAPK) and ERK2 (also known as MAPK1 or p42 MAPK) (Crews et al., 1992). ERK1/2 are activated by sequential phosphorylation of their regulatory tyrosine and threonine residues in the Thr-Glu-Tyr domain (Haystead et al., 1992). ERK, like Raf a serine/threonine kinase, further phosphorylates and activates more than 200 different substrates localized mainly in the nucleus but also in the cytoplasm or in other cellular organelles (Yao and Seger, 2009; Yoon and Seger, 2006). Among the cytosolic targets are kinases, phosphatases, cytoskeletal and signaling proteins, involved in processes like
7
3. Introduction translation, mitosis, and apoptosis. However, the main ERK1/2 targets are transcription factors and other transcriptional regulators (see list in (Yoon and Seger, 2006)). Target cytoplasmic proteins or transcription factors can be regulated directly by ERK but also by ERK-regulated MAPKAPKs (=Mitogen-activated protein kinase-activated protein kinases), such as ribosomal S6 kinases (RSKs), MAPK interacting kinases (MNKs), and mitogen and stress-activated kinases (MSKs). ERK, in order to phosphorylate and activate nuclear transcription factors, or transcription factors that have already been phosphorylated by ERK or ERK target kinases in the cytoplasm are translocated into the nucleus. Thus, nuclear ERK regulates a great variety of transcription factors, including c-Jun and c-Fos, which are part of the heterodimeric transcription factor AP-1 (activator protein 1), NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells), c-Myc (avian myelocytomatosis virus oncogene cellular homolog), and several family members of the Ets (E-twenty six) transcription factor family, especially Ets1 and Elk-1 (Chang et al., 2003). In this manner, ERK is involved in chromatin remodeling, in nuclear disintegration in mitosis, and in induction or suppression of transcription (Yao and Seger, 2009; Yoon and Seger, 2006).
3.1.2. Ras/ERK/MAPK Pathway Dysregulation – Causes of Cancer and Rasopathies
Ras/ERK/MAPK signaling has to be strictly regulated because upregulation of this pathway has been linked to malignant phenotypes including increased cell proliferation, migration, metastasis and invasiveness, defects in apoptosis, and the ability to induce neovascularization in tumors. Constitutive active mutants of Ras or B-Raf occur at a high frequency in almost all human cancers including pancreas, thyroid, lung, liver, kidney, bladder, breast, colorectal, gastric, and ovarian carcinoma, melanoma, seminoma, and leukaemia (Davies et al., 2002; Dhillon et al., 2007; Hilger et al., 2002; Malumbres and Barbacid, 2003; Montagut and Settleman, 2009). Thus, the activation of the Ras/ERK/MAPK pathway has to be strictly regulated because uncontrolled proliferation is a necessary step for the development of all cancers. Overactivation of the Ras/ERK/MAPK pathway is also causative for a growing group of diseases bundled as “rasopathies”. They are mainly caused by constitutive active mutants of Ras or Raf, but also by mutations of other pathway components or regulators that lead to upregulation of Ras-dependent signaling. All rasopathy-syndromes profoundly impact the development of various tissues and exhibit overlapping phenotypical features including facial dysmorphologies, cardiac defects, cutaneous abnormalities, neurocognitive delay, and a predisposition to malignancies (Tidyman and Rauen, 2009).
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3. Introduction
A variety of drugs are able to target and inhibit selectively Ras/Raf/ERK pathway components, e.g. Ras farnesyl transferase, Raf, MEK, and tyrosine kinase inhibitors (Dhillon et al., 2007; Hilger et al., 2002; Montagut and Settleman, 2009). In healthy species, of course, the Ras/Raf/ERK pathway also exhibits intrinsic regulation mechanisms. As shown above, the pathway comprises a set of protein kinases that are activated one by one and thereby transmit signals from receptor to nucleus. The activity of these kinases is regulated by sequential phosphorylation, autoregulation, protein phosphatases, and negative feedback regulation by pathway components or by independent regulators induced by cascade activation. Examples for such independent regulators are the SPRED proteins, which have been shown to potently inhibit the Ras/ERK/MAPK cascade in response to a wide range of mitogenic stimuli.
3.2. SPREDs – Inhibitors of the Ras/ERK/MAPK Pathway
The SPRED (Sprouty-related protein with EVH1 domain) protein family was detected twelve years ago in a yeast two-hybrid screen by their interaction with the tyrosine kinase domain of c- Kit. Together with the structural related Sprouty proteins, SPREDs represent a family of MAPK signaling inhibitors (Wakioka et al., 2001).
3.2.1. SPRED Isoforms
SPRED has first been identified in Drosophila melanogaster, where it is expressed in photoreceptor cells. The Drosophila orthologue AE33 therefore seems to play a role in the differentiation of photoreceptor cells during the development of the Drosophila eye (DeMille et al., 1996; Freeman et al., 1992; Treisman and Rubin, 1996). SPREDs seem to be ubiquitously expressed in most animal species, and besides the Drosophila AE33, two Xenopus tropicalis, three murine, and three human SPREDs have been described (Kato et al., 2003; Sivak et al., 2005; Wakioka et al., 2001). In mammalians, all three Spred genes are located on different chromosomes: In mice, Spred1 is located on 2E5 and in humans on 15q14, Spred2 on 11A3 and on 2p14, and Spred3 on 7B1 and 19q13, respectively ((Kato et al., 2003) and ENSEMBL database). Thus, in mice and men, the SPRED family consists of three members, namely SPRED1, SPRED2, and SPRED3. There is also first evidence for existing splice variants, e.g. Eve-3 (EVH1 enhanced), a splice variant of human SPRED3 (King et al., 2006).
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3. Introduction
3.2.2. SPRED Domain Structure and Functions
SPRED family members show a characteristic domain structure consisting of an N-terminal Enabled (Ena)/Vasodilator-stimulated phosphoprotein (VASP) homology 1 (EVH1) domain, a central c-Kit binding domain (KBD), and a C-terminal Sprouty-related (SPR) domain (Figure 2) (Kato et al., 2003; Wakioka et al., 2001). SPRED3 lacks a functional KBD (Kato et al., 2003), and its splice variant Eve-3 consists of merely a single EVH1 domain (King et al., 2006). The Spred2 mouse gene contains 1233 protein-coding nucleotides and the corresponding protein has a calculated molecular mass of 46 kDa. It comprises six exons, of which exons 1-3 encode the EVH1 domain, exons 4 and 5 encode the region between EVH1 domian and KBD, and exon 6 encodes the KBD and the SPR domain (Figure 2) (ENSEMBL Transcript ID: ENSMUST00000093298). The genomic structure of murine Spred1 (1335 bp coding sequence/49 kDa) is very similar, but it contains one more exon than Spred2, which encodes a part of the EVH1/KBD interregion (ENSEMBL Transcript ID: ENSMUST00000028829) (Bundschu, 2005; Bundschu et al., 2005). With six exons, Spred3 (1227 bp coding sequence/45 kDa) has a similar genomic arrangement like Spred2 but lacks a functional KBD domain due to the replacement of a critical arginine by a glycin (ENSEMBL Transcript ID: ENSMUST000000489239) (Kato et al., 2003).
Figure 2: SPRED2 Protein Structure and its Corresponding Genomic Exon/Intron Organization Exons (Ex) 1, 2, and 3 of Spred2 are coding for the EVH1 domain, exons 4 and 5 for the middle part, and exon 6 for both the KBD and the SPR domain. Numbers above the exons represent the 1233 protein-coding nucleotides of the Spred2 cDNA, which are translated into the 411 amino acid-containing SPRED2 protein. Figure based on (Bundschu, 2005).
3.2.2.1 The EVH1 Domain
The EVH1 domain is an evolutionary conserved protein domain containing about 115 amino acids. It represents a protein interaction module, which recognizes and binds specifically to proline-rich sequences (Ball et al., 2002; Niebuhr et al., 1997). To date, four classes of proteins that contain EVH1 domains have been identified, namely the Ena/VASP proteins, the Homer/Vesl proteins, the Wiskott-Aldrich syndrome proteins (WASP), and the SPRED proteins. 10
3. Introduction
Their common EVH1 domain is implicated e.g. in the regulation and remodeling of the actin cytoskeleton and cell motility (Gertler et al., 1996; Reinhard et al., 2001; Reinhard et al., 1995; Reinhard et al., 1996; Renfranz and Beckerle, 2002; Samarin et al., 2003), in axonal guidance (Menzies et al., 2004), in synaptic signal transduction (Sala et al., 2001; Xiao et al., 2000), and in cell proliferation and differentiation (Bundschu et al., 2006b, 2007). High-resolution structures of the EVH1 domains of all four protein classes were solved (Ball et al., 2002; Callebaut et al., 1998). However, the EVH1 domain of SPREDs shows a narrower peptide-binding groove indicating that it binds to less proline-rich sequences as compared to its related domains (Harmer et al., 2005; Zimmermann et al., 2004). Indeed, NBR1 (neighbor of BRCA1=breast cancer1) and NF1 (neurofibromin1), the only two identified proteins that interact with SPRED by binding to the EVH1 domain; show no canonical proline-rich binding sequences in their critical SPRED-interacting regions (Mardakheh et al., 2009; Stowe et al., 2012). The EVH1 domain has shown to be essential for SPRED protein function because N-terminal deletion mutants of SPRED1 and SPRED2 lost their ability to suppress ERK phosphorylation in response to different stimuli (King et al., 2005; Stowe et al., 2012; Wakioka et al., 2001). Vice versa, Eve-3 harboring exclusively the EVH1 domain is fully potent in inhibiting GF-induced MAPK signaling and cell cycle progression (King et al., 2006). Exceptionally, ∆N-SPRED2 is still functional in the suppression of hematopoietic cell differentiation in aorta-gonad-mesonephro- cultures (Nobuhisa et al., 2004) indicating a differential regulation of EVH1 function in the different SPRED isoforms, which is dependent on the cellular and mechanistic background.
3.2.2.2 The c‐Kit Binding Domain
The KBD represents a binding sequence for the oncogenic receptor tyrosine kinase c-Kit located in the central region of SPRED1 and SPRED2, and comprises approximately 50 amino acids. It is not related to any previously identified tyrosine kinase interaction domain such as SH2, PTB, or c-Met binding domain (Wakioka et al., 2001). The KBD seems to be involved but not essential for SPRED protein function. Thus, SPRED3 lacking the KBD as well as ∆KBD- SPRED1 mutants are still able to suppress GF-induced ERK phosphorylation, although inhibitory potential is reduced (Kato et al., 2003; Nonami et al., 2004; Wakioka et al., 2001).
3.2.2.3. The Sprouty Domain
SPREDs contain a highly-conserved cysteine-rich Sprouty-related domain at their C-terminus consisting of about 110 amino acids. As the name implies, this SPR domain is homologous to the C-terminus of Sprouty proteins comprising a family of four homologous mammalian
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3. Introduction members (de Maximy et al., 1999). Both in Drosophila and in vertebrates, Sproutys, similar to SPREDs, have been described as negative regulators of MAPK signaling downstream of a variety of RTKs (Casci et al., 1999; Hacohen et al., 1998; Kim and Bar-Sagi, 2004; Kramer et al., 1999; Reich et al., 1999). In SPRED2, the SPR domain is essential and sufficient for MAPK pathway inhibition (King et al., 2005; Nobuhisa et al., 2004; Sasaki et al., 2003), whereas in SPRED1 it is not required to inhibit ERK phosphorylation in response to various stimuli (King et al., 2005). ∆C-SPRED2 has been shown to fail to inhibit Ras/ERK/MAPK signaling and to even enhance it by exerting a dominant negative effect on SPRED function (King et al., 2005). Although ∆C-SPRED1 seems to retain the inhibitory potential (King et al., 2005), contrarily a dominant negative effect on MAPK activation has also been published (Nonami et al., 2004; Wakioka et al., 2001). Endogenous and functional SPRED is localized in vesicle-like structures (Engelhardt et al., 2004; Mardakheh et al., 2009; Phoenix and Temple, 2010) at the plasma membrane, where it exerts its inhibitory effect on possible MAPK pathway targets, like Ras or Raf, which are also recruited to the membrane upon activation (King et al., 2005; Wakioka et al., 2001). However, ∆C-SPREDs localize either in the cytoplasm (Kato et al., 2003; Stowe et al., 2012; Wakioka et al., 2001) or in the nucleus (Mardakheh et al., 2009). Therefore, according to Sproutys, the SPR domain is essential for the membrane anchoring of SPREDs and targets them to phosphatidylinositol 4,5-bisphosphate in plasma membranes upon MAPK pathway stimulation (Kato et al., 2003; Lim et al., 2002; Wakioka et al., 2001). Additionally, SPRED1 and SPRED2 are able to form stable heterodimers mediated exclusively by their SPR domains (King et al., 2005). Taken together, the biochemical inhibitory function of SPREDs on the Ras/ERK/MAPK pathway is thought to reside mainly in the EVH1 domain, whereas the SPR domain mostly mediates the protein localization to cell membranes and the association with interaction partners, all required for appropriate protein function.
3.2.3. SPRED Expression Patterns and Localizations
To date, SPRED expression has been investigated well in humans, rats, and mice by monitoring endogenous Spred promoter activity and by visualizing expression on RNA and protein levels (Figure 3). Investigation of especially Spred2 promoter activity was enabled using SPRED2 knockout (KO) mice generated by an insertion of a gene trap vector-encoded reporter gene into the Spred2 gene (Figure 12) (Bundschu et al., 2006a; Ullrich and Schuh, 2009). During mouse embryongenesis from day E7.5 to E13.5 the endogenous Spred2 promoter was highly active in ectodermal and mesodermal tissues in early stages and later in developing 12
3. Introduction neural tissues, heart, lung, intestine, urogenital tract, and limbs (Figure 3 A) (Tuduce et al., 2010). In newborn and adult SPRED2 KO mice, Spred2 promoter activity was nearly congruent and similarly ubiquitous compared to the embryos. The highest activity was detected in various parts of the brain, in the telencephalon especially in frontoparietal cortex and hippocampus, and in the cerebellum mostly in cortex and Purkinje cells. A comparable highly activated promoter was also seen in other neural tissues like spinal cord and spinal nerves. A strong activity was observed in glands like salivary gland, prostate and mucus epithelial glands in stomach and colon, in the kidney, especially in the cortex, and in vascular, intestinal, and uterine smooth muscle cells. Weaker activity was detected in heart, lung, liver, bones, and testis and almost no activity was seen in skeletal muscle and spleen (Bundschu et al., 2006a).
Figure 3: Ubiquitous Spred2 Promoter Activity and SPRED2 Expression in Embryonic and Adult Mice A) Lateral right view of an X-Gal-stained whole mount SPRED2 HET embryo at day E11.5 demonstrating Spred2 promoter activity by blue staining. 1: Mesencephalon, 2: otic vesicle, 3: optic vesicle, 4: liver, 5: hindlimb bud, 6: forelimb bud, 7: somites, 8: neuroepithelium of neural tube, 9: maxillar and mandibular component of first branchial arch, 10: rhombencephalon. Figure in part reproduced from (Tuduce et al., 2010) with permission from Wiley. B) RT- PCR of different tissues of adult mice showing widespread SPRED2 expression on mRNA level. Figure in part reprinted from (Engelhardt et al., 2004) with permission from Spriger.
Determination of Spred2 RNA levels by RT-PCR (Figure 3 B) and Northern blot, and of SPRED2 protein levels by Western blot again confirmed the ubiquitous expression pattern of SPRED2 in mainly brain, glands, and kidney (Engelhardt et al., 2004; Kato et al., 2003). In contrast to the ubiquitous SPRED2 expression, murine SPRED1 expression has predominantly been detected in brain tissues, and SPRED3 expression exclusively in brain, as shown by RT- PCR, Northern blot, and Western blot (Engelhardt et al., 2004; Kato et al., 2003). In situ hybridisation of fetal rat tissue demonstrated a similar SPRED1 and SPRED2 expression in rat with high levels of RNA transcripts in brain, spinal cord, lung, heart, intestine, and skin (Hashimoto et al., 2002). 13
3. Introduction
Immunohistochemistry with human tissue revealed high SPRED2 protein levels in glandular tissues like salivary glands, prostate, and sweat glands, which is consistent with high glandular expression also seen in mice. There, SPRED2 especially localized in vesicle-like granules indicated by colocalization with specific endosome markers (Engelhardt et al., 2004). Taken together, SPRED2 shows a widespread expression pattern in men and mice, with varying intensity depending on the regarded tissue but present from early embryonic stages up to adulthood.
3.2.4. SPREDs and Suppression of Ras/ERK/MAPK Signaling
SPREDs suppress Ras/ERK/MAPK pathway activity downstream of multiple RTKs (e.g. FGFR, EGFR, VEGFR, PDGFR, NGFR) in response to a wide range of mitogenic stimuli like different growth factors (e.g. FGF, EGF, VEGF, PDGF, NGF, HGF, SCF), serum, cytokines (e.g. ILs), and chemokines (Bundschu et al., 2005; Inoue et al., 2005; Kato et al., 2003; King et al., 2005; Miyoshi et al., 2004; Nobuhisa et al., 2004; Nonami et al., 2004; Nonami et al., 2005; Sasaki et al., 2003; Sasaki et al., 2001; Sivak et al., 2005; Wakioka et al., 2001), for review see (Bundschu et al., 2007). The inhibitory effect is mediated by interaction with defined pathway components at different positions of the Ras/ERK/MAPK pathway. Caveolin1, not a direct participating component in the Ras/ERK/MAPK pathway but a designated regulator of signal transduction, is localized in caveolae. In these lipid-rich invaginations of the plasma membrane it associates with SPRED1 and other pathway components like EGFR (Couet et al., 1997) or Ras (Song et al., 1996). The interaction of Caveolin1 with the SPR domain of SPRED1 increases the inhibitory effect of SPRED1 on ERK phosphorylation; however, the underlying mechanism remains to be shown (Nonami et al., 2005). Under unstimulated conditions, SPREDs seem to be constitutively associated with RTKs. Thus, via the SPR domain, SPRED1 is bound to FGFR-like 1 (FGFRL1) (Zhuang et al., 2011), and SPRED2 to EGFR from where it dissociates upon EGF stimulation in order to exert its suppressive effect (Meng et al., 2012). The RTKs itself can be a target of SPREDs inhibitory function because EVH1 domain–mediated interaction of SPRED2 with NBR1 induces lysosomal degradation of the FGFR, thus silencing FGF-dependent ERK activation (Mardakheh et al., 2009). Via the conserved Raf-binding motif (RBM) within the SPR domain, SPREDs as well as Sproutys are able to interact with the C-terminal catalytical domain of Raf1 (Sasaki et al., 2003). The small GTPase Ras (Wakioka et al., 2001) acting upstream of Raf kinase in the MAPK pathway also binds to SPREDs, but the binding sites have yet to be identified. Accordingly, one 14
3. Introduction possible SPRED target has been shown to be located between Ras and Raf (Figure 4). SPRED is able to interact with Ras in the Ras-Raf complex, but does not prevent the activation of Ras. However, although Ras is still capable of recruiting Raf to the membrane, Raf is thought to be not accessible to its kinase when SPRED is bound to the Ras-Raf complex (Wakioka et al., 2001). SPRED has further been shown to decrease Ras-GTP levels, possibly by inhibiting RasGEF or enhancing RasGAP activity, and thus the second identified SPRED target lies upstream of Ras (King et al., 2005) (Figure 4). Indeed, SPREDs have been shown to interact with the RasGAP NF1 via their EVH1 domain. SPRED1 enables and enhances the RasGAP activity of NF1 by recruiting it to the plasma membrane, where it accelerates the hydrolysis of active Ras-GTP to the inactive Ras-GDP form (Stowe et al., 2012).
Figure 4: SPREDs – Inhibitors of the Ras/ERK/MAPK Cascade SPREDs suppress Ras/ERK/MAPK signaling at different sites of action. Possible SPRED targets have been described to be located upstream of Raf or between Ras and Raf (CP = cytoplasmic proteins). Figure based on (Bundschu, 2005).
In sum, point and mechanism of Ras/ERK/MAPK pathway regulation by SPREDs seems to be variable dependent on cell type, tissue or developmental process to be regulated. According to current knowledge, SPREDs do not affect other cellular pathways like e.g. JAK–STAT, PLC, JNK, and p38MAPK pathways. Thus, the suppressive effect of SPREDs seems to be exclusively restricted to Ras/Raf/ERK signaling (King et al., 2006; King et al., 2005; Miyoshi et al., 2004; Nobuhisa et al., 2004; Nonami et al., 2004; Sivak et al., 2005; Wakioka et al., 2001).
3.2.5. Regulation of SPRED‐mediated Ras/ERK/MAPK Pathway Inhibition
Protein expression, activity, and function can be regulated by pretranslational or posttranslational processes. Pretranslational alternative splicing takes place within the SPRED
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3. Introduction family and has been demonstrated by Eve-3, a liver specific splice variant of SPRED3. The functional EVH1 domain conserves its inhibitory effect on MAPK signaling, whilst the loss of the SPR domain causes an altered localization pattern in cytoplasmic punctae rather than at the cell membrane (King et al., 2006). Besides Eve-3, a variety of further splice mutants may exist (see ENSEMBL database and (Ullrich et al., 2011)). SPRED expression can also be regulated pretranslationally by microRNAs (miRs), short non- coding RNAs that function as negative regulators of gene expression. Thus, SPREDs and their inhibitory function on Ras/ERK/MAPK signaling is downregulated by miRs like miR-126 (Fish et al., 2008; Wang et al., 2008), miR-221, and miR-485-3p (Liu et al., 2012). Among the posttranslational modifications, phosphorylation and ubiquitination have been shown to be critical for SPRED regulation. Murine SPRED1 and 2 are tyrosine phosphorylated in response to stimulation by various growth factors (Wakioka et al., 2001). c-Kit may be critical for SCF-induced tyrosine phosphorylation because the KBD is required and because SPRED3 without a functional KBD can not be tyrosine phosphorylated (Kato et al., 2003; Nonami et al., 2004; Wakioka et al., 2001). Two recent studies identified defined tyrosine phosphorylation sites in murine SPREDs, which are critical for their inhibitory effect. Thus, Y377 and Y420 in the SPR domain of mouse SPRED1 and 2 have to be phosphorylated for mediating Ras/ERK/MAPK pathway suppression. Vice versa, interaction of the protein tyrosine phosphatase SHP2 with the EVH1/KBD interregion of SPRED1 and 2 causes dephosphorylation of Y420, hence attenuating SPRED-mediated pathway inhibition (Quintanar-Audelo et al., 2011). Similarly, phosphorylation of Y303, Y343, and Y353 within SPR of SPRED2 seems to trigger its dissociation from the EGFR upon EGF stimulation and is essential for the interaction of SPRED2 with the p85 subunit of phosphatidylinositol 3 kinase. This interaction in turn is thought to enhance the SPRED2- mediated inhibitory effect via increased Ras binding to SPRED2 and to decrease SPRED2 ubiquitination (Meng et al., 2012). In humans, comparable tyrosine phosphorylation required for the inhibitory effect of SPREDs could not yet be detected; however, phosphorylation of serine and threonine residues has been demonstrated in human SPREDs (King et al., 2005). Ubiquitination is a posttranslational enzymatic process, by which ubiquitin is ligated to a target protein and thus labels it for degradation. It can also be regulated by phosphorylation, whereas phosphorylation of the above mentioned Y303, Y343, and Y353 in SPR domain of SPRED2 seems to prevent SPRED degradation (Meng et al., 2012). However, phosphorylation of Y228 and/or Y231 in the c-Kit binding domain of SPRED2 is required for the interaction with Cbl (Casitas B-lineage lymphoma) E3 ubiquitin ligase and thus enhances SPRED ubiquitination (Lock et al., 2006). In sum, a combination of differential splicing, miRs, phosphorylation, and ubiquitination enables a fine tuning of SPRED functions and expression levels.
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3.2.6. SPRED Functions in Mice and Men
SPRED proteins are inhibitors of Ras/ERK/MAPK signaling, a pathway that regulates cell proliferation and differentiation in a tremendous variety of cell types, from unicellular eukaryotes over fungi and plants up to all animals, from early development up to old age. ERK is activated by multiple RTKs in response to an even greater diversity of ligands and itself activates an immense amount of nuclear and cytoplasmic target proteins. Due to their involvement in this highly conserved and ubiquitous pathway, SPREDs play a role in a great variety of developmental processes.
3.2.6.1. In vitro Studies and Mouse Models
To date, five different mouse models are available to study Spred1 and Spred2 gene function: First, one SPRED1 (Denayer et al., 2008; Inoue et al., 2005; Nonami et al., 2004) and one SPRED2 KO mouse model (Nobuhisa et al., 2004; Wakabayashi et al., 2012) both backcrossed to C57BL/6 background and generated by a Japanese group. Second, one knockdown mouse line for SPRED1 (Phoenix and Temple, 2010). Third, the SPRED2 KO mouse model generated by us and kept on a mixed C57BL/6 x 129P2/OlaHsd background (Bundschu et al., 2005; Ullrich et al., 2011).
Hematopoiesis The first function that has been described for SPREDs is its role as a regulator of hematopoiesis. In mouse embryos, SPRED2 is expressed in the AGM (aorta-gonad- mesonephros) region, a primary site of embryonic hematopoiesis. In SPRED2 KO mice the production of CD45+ hematopoietic stem cells and colony formation is upregulated compared to WT controls. Accordingly, overexpression of SPRED2 significantly reduces ERK phosphorylation and the number of CD45+ cells in an AGM culture (Nobuhisa et al., 2004). SPRED1 is highly expressed in mature hematopoietic cells. Thus, in different IL-3-dependent hematopoietic cell lines, overexpression of SPRED1 reduces ERK activation, cell proliferation and colony formation. Vice versa, in bone marrow-derived mast cells of adult SPRED1-deficient mice, Ras/ERK/MAPK signaling and proliferation was augmented (Nonami et al., 2004). In an allergic asthma model, adult SPRED KO mice develop airway eosinophilia and hyperresponsiveness, indicating that SPRED1 also downregulates IL-5-induced Ras/ERK/MAPK pathway activation required for eosinophil production (Inoue et al., 2005). Taken together, due to their suppressive effect on ERK-dependent cell proliferation and
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3. Introduction differentiation, SPRED2 negatively regulates embryonic hematopoiesis, while SPRED1 rather regulates late-phase hematopoiesis by inhibiting hematopoietic cytokine signaling.
Angiogenesis and Lymphangiogenesis Angiogenesis is mainly regulated by GFs like FGF and VEGF, which stimulate MAPK pathways that mediate the generation of new vessels by inducing escape of endothelial cells from their original blood vessel, proliferation, and sprouting. Recently, miRNAs have been identified as novel pretranslational regulators of angiogenesis (Suarez and Sessa, 2009). SPRED1 was shown to be a target of the miR-126, expressed specifically in endothelial cells and regulating VEGF-mediated angiogenesis by repression of target genes. miR-126 deficiency in mice (Wang et al., 2008) and knockdown in zebrafish (Fish et al., 2008) resulted in loss of vascular integrity, hemorrhage, and partial embryonic lethality. Both studies demonstrate that ablation of miR-126 normally repressing SPRED1 expression leads to a decrease in ERK signaling in response to VEGF. Thus, the resulting impaired endothelial cell proliferation, migration, and angiogenesis is a consequence of forced SPRED1-mediated inhibition of angiogenic signaling (Fish et al., 2008; Wang et al., 2008). Lymphangiogenesis is mediated in part also by VEGF/ERK-dependent signaling and regulates the growth of lymphatic vessels form pre-existing vessels. SPRED1 and SPRED2 seem to play an important role in lymphangiogenesis because their deficiency in mice results in embryonic lethality due to severe subcutaneous hemorrhage, edema, and dilated lymphatic vessels filled with erythrocytes. These SPRED1/2-double KO mice show increased numbers of lymphatic vessels and resemble mouse models defective in the separation of lymphatic vessels from blood vessels. Because VEGF-C-mediated ERK activation was enhanced in SPRED1/2-double KO embryos, SPREDs seem to be inhibitors of VEGF-induced lymphatic endothelial cell proliferation (Taniguchi et al., 2007).
Brain Development and Learning All SPRED proteins show a high expression pattern in brain and spinal cord, which is indicative for their functional requirement there. Indeed, in fetal mouse brain, SPRED1 was shown to be highly enriched in central nervous system (CNS) germinal zones during neurogenesis, especially in the ventricular and subventricular zone of the cortex, where neural progenitor cells (NPC) reside. Knockdown of SPRED1 in vitro in embryonic cortical cells revealed increased ERK phosphorylation, elevated self-renewal, and proliferation in NPCs. In vivo knockdown of SPRED1 in mouse embryos also leads to an increase in NPC proliferation and periventricular heterotopia, a developmental abnormality characterized by a mislocalization of cell masses in or next to the subventricular zone due to abnormal neuronal migration. Thus, SPRED is critical for
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3. Introduction normal cortical development and, by controlling MAPK/ERK-dependent NPC proliferation, helps to maintain the integrity of germinal zones (Phoenix and Temple, 2010). Besides their role in brain development, SPREDs are also critical for proper brain function. Behavioral characterization of a SPRED1 KO mouse line revealed unaltered neuromotor performance and normal sensory perception to visual, auditory, or pain stimuli but an impaired spatial and visual learning and memory. In this manner, the ability to find and remember the position of a dry platform in a swimming pool (Morris water maze) or to discriminate between black and white colored doors and remember the color of the door with a reward behind in a T- shaped land maze (T-maze) was impaired in SPRED1 KO mice. Moreover, electrophysiological recordings on hippocampal slices revealed reduced excitatory postsynaptic potentials (EPSPs) and defects in short and long term synaptic plasticity, both underlining the observed deficits in hippocampus-dependent learning and memory formation. These deficits seem to be related to hyperactivation of the Ras/ERK/MAPK pathway because after induction of long term potentiation, ERK phosphorylation was increased in brain slices of SPRED1 KO mice (Denayer et al., 2008). DYRK1A (dual specificity tyrosine-phosphorylation-regulated kinase 1A), a kinase thought to play a role in Down syndrome, shows overlapping expression patterns with SPREDs in the brain and has been identified as a new SPRED1/2 binding partner. The interaction of SPREDs via their SPR domain with the kinase domain of DYRK1A prevents it from phosphorylating its targets like STAT3 and Tau. By competing with DYRK1A substrates for binding and without affecting its kinase activity, SPREDs are able to inhibit DYRK1A-mediated cell proliferation and, as DYRK1A regulators, may impact Down syndrome development (Li et al., 2010).
Embryonic Development and Growth FGF/FGFR signaling plays a critical role in embryonic development including mesoderm induction, anterior-posterior patterning, neural induction, and limb development. SPREDs are highly expressed already during embryonic development especially in ectodermal and mesodermal tissues (Tuduce et al., 2010) and are critical regulators of FGF-dependent cell proliferation. During gastrulation of Xenopus tropicalis, XtSPREDs inhibit mesoderm specification by inhibiting FGF-dependent MAPK activation (Sivak et al., 2005). In the pancreas mesenchyme of the embryonic mouse, SPRED1 expression is stimulated by FGF-9 signaling (Sylvestersen et al., 2011). In the lung mesenchyme of embryonic rats, SPREDs are also coexpressed with FGF-9 and-10 and seem to be essential for normal lung branching (Hashimoto et al., 2002). Hepatospecific Eve-3 regulates the regenerative capacity, growth, and mass of the mature liver by suppressing ERK/MAPK singnaling (King et al., 2006).
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Figure 5: SPRED2 Deficiency in Mice Causes Dwarfism SPRED2 KO mice have a short stature and decreased long bone growth already from birth on, which is similar to hypochondroplasia in humans.
FGFR pathways are also critical regulators of bone development, especially FGFR3-mediated signaling, which suppresses long bone growth. Thus, FGFR3 gain-of-function mutations leading to increased ERK/MAPK signaling result in dwarfism. Accordingly, SPRED2 deficiency in mice provoked hypochondroplasia-like dwarfism (Figure 5) associated with reduced growth and body weight, shortened bones, and narrowed growth plates. Because FGF-stimulated chondrocytes of SPRED2 KO mice showed earlier and augmented ERK phosphorylation, SPRED2 regulates long bone growth by suppression of FGF-dependent ERK activation (Bundschu et al., 2005).
Cytoskeleton and Vesicular Trafficking MAPK signaling is not only a critical regulator of cell proliferation and differentiation but also impacts cell motility. Similar to the related VASP, SPREDs have been shown to play a role in the reorganization of the actin cytoskeleton. SPREDs are able to directly interact with RhoA (Ras homolog gene family, member A) and to inhibit Rho-mediated ROCK (Rho-associated protein kinase) activation, actin stress fiber formation, and cell migration (Miyoshi et al., 2004). A second SPRED target in the RhoA/ROCK pathway is TESK1 (testis-specific protein kinase 1), a downstream target of ROCK that modulates actin reorganization via phosphorylation of cofilin. While unphosphorylated and active cofilin induces disassembly of actin filaments, phosphorylated and inactive cofilin induces stress fiber formation. SPRED1 inhibits TESK1 by SPR domain-dependent interaction and prevents TESK1 to phosphorylate cofilin. The resulting stress fiber breakdown renders the actin network dynamic (Johne et al., 2008). Via its SPR domain, SPRED1 has further been shown to interact with MARKK (microtubule affinity-regulating kinase-activating kinase), which causes the breakdown of microtubules by phosphorylation and activation of downstream targets. However, the functional impact of this interaction has yet to be identified (Johne et al., 2008). Associated with a putative role of SPREDs in microtubule dynamics, there are some first hints that SPREDs might play a role in microtubule-guided vesicular trafficking. SPRED2 colocalizes with NBR1 in cytoplasmic punctae representing late endosomes as identified by costaining with Ras-related protein (Rab) 7. Via interaction with SPRED2, NBR1 indirectly enhances SPREDs 20
3. Introduction inhibitory effect on ERK phosphorylation. SPRED2-NBR1 induces the targeting activated FGFRs to late endosomes for lysosomal degradation (Mardakheh et al., 2009). Similarly, the periventricular heterotopia characterized by CNS germinal zone disruption occurring in SPRED1 knockdown mice is reminiscent of mouse models harboring mutations of genes associated with vesicular trafficking (Phoenix and Temple, 2010). Rab-GTPases regulate many steps of membrane traffic, including vesicle formation, vesicle movement, and membrane fusion. Colocalization of SPREDs has been demonstrated not only with the late endosome markers Rab7 (Mardakheh et al., 2009), but also with Rab5 and Rab11 in transfected HEK 293 T cells (Engelhardt et al., 2004) and in NPCs of mouse embryos (Phoenix and Temple, 2010). Whereas SPRED1 seems to extensively colocalize with early endosome marker Rab5, but to a lesser extent with the recycling endosome marker Rab11 (Phoenix and Temple, 2010), SPRED2 rather colocalizes with Rab11 than with Rab5 (Engelhardt et al., 2004).
Tumorigenesis and Metastasis SPREDs, arising from their function as negative regulators of Ras/ERK/MAPK signaling, a main regulator of cell proliferation and differentiation, have a putative role in preventing the development of tumors and metastasis. In this manner, an in vitro study with Hela cells, in which large tumor suppressors (LATS) 1 and 2 were knocked down, show increased proliferation and migration associated with dysregulated expression of various oncogenes, hereunder SPRED1, which was downregulated (Visser and Yang, 2010). Similarly, in a tumorigenic murine keratinocyte cell line (PDV), endogenous SPRED2 expression was decreased compared with a non-tumorigenic control. However, stable expression of SPRED2 in these PDV cells suppresses transforming growth factor-β1 (TGF-β1)-induced ERK activation, urokinase type plasminogen activator (uPA) expression, epithelial mesenchymal transition, and cell migration, all contributing to tumor invasion and metastasis (Villar et al., 2010). The anti-tumorigenic and anti-metastatic effect of SPREDs could also be confirmed by in vivo studies in different mouse models. In the highly metastatic mouse osteosarcoma cell line LM8, SPRED1 expression inhibits ERK activation, proliferation, and migration in vitro. Accordingly, in nude mice injected with LM8 cells overexpressing SPRED1, LM8 cell-mediated tumor growth and metastasis was suppressed (Miyoshi et al., 2004). SPRED1 overexpression also reduces tumorigenesis in a hepatocellular carcinoma (HCC) model in nude mice. Accordingly, in cultured HCC cells, overexpression of SPRED1 suppresses cell proliferation, ERK activation, cell motility, and the expression of matrix metalloproteinase (MMP) 2 and 9, which are required for extracellular matrix degradation and metastasis (Yoshida et al., 2006). The same effect on HCC was shown for SPRED2, both in HCC cultures and in a nude mouse model, with the additional
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3. Introduction outcome that SPRED2 overexpression induces the activation of caspase-3 and apoptosis (Ma et al., 2011). Similarly, SPRED2 seems to have a tumor-suppressive effect on prostate cancer both in vivo and in vitro (Kachroo et al., 2013). Somewhat different, an immunological study with C57BL/6 mice injected with the murine colon adenocarcinoma cell line MC38 reveals TGF-β- induced impaired functionality of CD8+ cytotoxic T cells in the tumor microenvironment. This is characterized by hyporesponsiveness to cytokines, downregulation of TCR pathway components, reduced T cell proliferation, and decreased ERK activation, which is in this case contributed by upregulation of SPRED1 expression in the cytotoxic T cells (di Bari et al., 2009). SPRED expression levels seem to be further responsive to and coefficient with cancer pharmaceuticals. The anti-tumorgenic silibinin increases the expression of SPRED1/2 in a human hepatocellular carcinoma cell model (Hep G2) (Momeny et al., 2008). Furthermore, Imatinib, a common drug used for chronic myeloid leukemia (CML) treatment, promotes endogenous SPRED2 levels in the human CML cell line K562 (Liu et al., 2010).
3.2.6.2. First Lessons from Humans
Since the discovery of SPREDs and their identification as negative Ras/ERK/MAPK cascade regulators in 2001, there are now some clear indications of what a missing or altered SPRED expression causes in humans. While dysregulated human SPRED expression has been linked to the development of cancers, mutations in human SPRED1 leading to complete or partial deficiency are causative for rasopathies, a genetically related group of neuro-, cardio-, facio-, and cutaneous-developmental disorders.
Cancer To date, only two studies investigating the impact of SPREDs in human cancer have been published. In tumors of human HCC patients, ERK phosphorylation was increased, while expression of SPRED1/2 was downregulated. Interestingly, in HCC patients with low SPRED expression levels, the incidence of tumor invasion and metastasis was higher than in patients with higher SPRED levels, indicating an inverse correlation between SPRED expression and tumorigenesis (Yoshida et al., 2006). In prostate cancer tumors, SPRED2 was also downregulated, while SPRED1 expression remained unchanged, indicating a unique role for SPRED2 in prostate cancer (Kachroo et al., 2013). Although SPRED mutations have not yet been shown to be causative for any kind of cancer, they are, due to the observed downregulation in cancers and due to their anti-proliferative effect, prognostic factors and potential targets for anti-cancer and anti-metastatic therapies.
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3. Introduction
Legius Syndrome Just recently, Legius syndrome has been described as an autosomal dominant human disorder, which was detected due to its symptomatic similarity to neurofibromatosis type 1 (Brems et al., 2007). In a neurofibromatosis clinic, five families were identified at first, showing clinical features of neurofibromatosis type 1 but lacking any mutation in the neurofibromin gene NF1. A genome- wide linkeage scan revealed germline loss-of-function mutations in the Spred1 gene in these patients (Brems et al., 2007). Legius syndrome and neurofibromatosis type 1 belong to the family of rasopathies, of which the most prominent are Costello syndrome, Noonan syndrome, LEOPARD syndrome, and cardio-facio-cutaneous syndrome. All these diseases show overlapping phenotypes, originating from germline mutations in genes encoding a component or a regulator of the Ras/ERK/MAPK pathway and leading to pathway upregulation (Tidyman and Rauen, 2009). While neurofibromatosis type 1 and Legius syndrome are caused by loss-of- function mutations of the pathway inhibitors NF1, a RasGAP (Cawthon et al., 1990; Viskochil et al., 1990; Wallace et al., 1990), and SPRED1 (Brems et al., 2007), respectively, the other rasopathies are caused by gain-of-function mutations of direct Ras/ERK pathway components like PTPN11/SHP2 (Digilio et al., 2002; Legius et al., 2002; Tartaglia et al., 2001), Sos (Roberts et al., 2007; Tartaglia et al., 2007), H-Ras (Aoki et al., 2005), K-Ras (Niihori et al., 2006; Schubbert et al., 2006), C-Raf (Pandit et al., 2007; Razzaque et al., 2007), B-Raf (Niihori et al., 2006; Rodriguez-Viciana et al., 2006), and MEK1/2 (Rodriguez-Viciana et al., 2006). Until today, about 100 different Spred1 mutations have been described in about 150 unrelated individuals (Brems et al., 2007; Brems et al., 2012; Denayer et al., 2011; Laycock-van Spyk et al., 2011; Messiaen et al., 2009; Muram-Zborovski et al., 2010; Pasmant et al., 2009b; Spencer et al., 2011; Spurlock et al., 2009; Stevenson et al., 1993). Most of the described mutations are point mutations, including nonsense, missense, and frameshift mutations, but also in-frame and whole gene deletions as well as intronic mutations have been reported. The mutations affect both the EVH1 and the SPR domain, and in vitro experiments with subcloned in vivo detected Spred1 mutations revealed a reduced inhibitory effect on ERK activation regardless of the mutations domain localization (Brems et al., 2007). All known Spred1 gene variants are collected in the public and regularly updated SPRED1 database of ARUP Laboratories and the University of Utah: http://www.arup.utah.edu/database/SPRED1/SPRED1_welcome.php (Sumner et al., 2011). Legius syndrome in humans is characterized by cutaneous café au lait macules with or without axillary freckling, lipomas, learning disabilities, developmental delays, and macrocephaly (Brems et al., 2007; Brems et al., 2012). Very strikingly, SPRED1 KO mice also show learning disabilities (Denayer et al., 2008) and facial abnormalities (Inoue et al., 2005), and therefore reflect a demonstrable congruent phenotype with that of Legius syndrome patients. These
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3. Introduction phenotypical features of Legius syndrome are also characteristic for neurofibromatosis type 1. The additional predisposition to malignancies like tumors of the peripheral and central nervous system (neurofibromas, astromas, optic pathway gliomas), Lisch nodules, and osseus lesions in neurofibromatosis type 1 is completely absent in Legius syndrome (Brems et al., 2007; Messiaen et al., 2009; Pasmant et al., 2009a). Hence, genetic testing to clearly distinguish between neurofibromatosis type 1 and the milder Legius syndrome is indispensable for proper disease diagnosis and prognosis. Mutational analysis of Spred2 in Legius syndrome patients did not detect any mutations (Spurlock et al., 2009) and thus, a human correlate of SPRED2 deficiency remains to be identified.
3.3. The Mouse ‐ A Research Model for Human Gene Function and Disease
The human and mouse genomes were fully sequenced at the beginning of the new millennium (Lander et al., 2001; Venter et al., 2001; Waterston et al., 2002). Now, one major challenge facing the scientific community is to elucidate the in vivo function of the 20,000-25,000 identified protein-coding genes. Large-scale generation of genetically modified Drosophila, C. elegans, Xenopus, and zebrafish organisms has been carried out for many years. Although these non- mammalian animals have provided indispensable insights into gene function, the mouse is considered to be the most valuable model to study mammalian gene function. A great advantage of the mouse is its genetic similarity with humans because human and murine genes are homologous to 99% (Lander et al., 2001; Venter et al., 2001; Waterston et al., 2002). But also mouse development, physiology, anatomy, behavior, and diseases have much in common with those of humans due to their common ancestry. Although the same is true for rats, most techniques for genetic manipulation that result in gene knockouts, knockins, or in conditional mutations depend on the ability to culture and manipulate embryonic stem (ES) cells. Whereas the mouse genome supports modifications in ES cells, it was not possible for a long time to generate ES cells from rats with the same potential to provide pluripotency, genetic manipulation, and germ line transmission like in mice. ES cells have been available from inbred mice since 1981 (Evans and Kaufman, 1981; Martin, 1981); however, the first equivalent rat ES cells have been generated much later (Buehr et al., 2008; Li et al., 2008). Therefore, manipulating techniques not requiring ES cells, e.g. the generation of transgenic models by microinjection of DNA into fertilized oocytes, are feasible in both rats and mice according to the 24
3. Introduction experimental intent. However, for global or conditional knockout or knockin approaches involving ES cell manipulation and culture, the mouse is still the model of choice.
3.3.1. Mutagenesis Strategies in Mice
The use of mice as a model organism to study human diseases and to identify therapeutic targets by mutating or knocking out genes has a more than 100 year old history. It started with the collection of naturally occurring and visible mouse mutants. The development of mouse genetics to a formalized science enabled a more systematic collection and analysis of spontaneous mutants like agouti (Bultman et al., 1992), reeler (D'Arcangelo et al., 1995), and obese (Zhang et al., 1994) and facilitated their mapping to the mutated genes. Because the frequency at which spontaneous mutations occur is very low (~5 x 10-6 per locus), the first X-ray mutagenesis experiments were carried out in the 1930s (Russell et al., 1958), followed by studies with chemical mutagens like chlorambucil (Russell et al., 1989) and N-Ethyl-N- nitrosourea (ENU) (Russell et al., 1979). Whereas the mutagenesis frequency induced by radiation or chemical mutagens is 20-300 times greater than that of spontaneous occurring mutants, both X-ray and chlorambucil mutagenesis can cause chromosomal rearrangements. Therefore, on the one hand, they provide a landmark for identifying and cloning the affected gene(s), but on the other hand, these methods mostly affect multiple genes and thus impede the dissection of individual gene function. In contrast, ENU is easily administrable, amenable to high throughput, and the generated mutations are primarily single gene mutations (point mutations or small deletions not exceeding 50 bp). Hovewer, it does not provide obvious molecular landmarks for cloning. Since the late 1970s, insertional mutagenesis began to be hotly pursued in the mouse, and the possibility to directly introduce exogenous genetic material into the mouse embryo predates the widespread use of recombinant DNA techniques these days. In two independent studies, Rudolf Jaenisch et al. showed that viral DNA sequences can be integrated into the mouse genome both after microinjection into the blastocoel of mouse blastocysts and after viral infection of preimplantation mouse embryos, and that germline transmission is possible (Jaenisch, 1976; Jaenisch and Mintz, 1974). Shortly after, the first successful introduction of a cloned gene into the mouse genome was reported. Therefore, DNA was microinjected into the male pronucleus of a one-cell stage fertilized embryo, which was reimplantated into foster mothers (Gordon et al., 1980). In 1981, several groups were not only successful in introducing cloned genes by this technique, but also in germline transmission of these genes. Thus, the first transgenic mice were generated (Brinster et al., 1981; Costantini and Lacy, 1981; Gordon and Ruddle, 1981; Harbers et al., 1981; Wagner et al., 1981a; Wagner et al., 1981b). Transgenic mice mainly 25
3. Introduction represent gain-of-function mutations. Their most common use is on the one hand the study of tissue- and developmental-specific gene regulation, on the other hand the research of the phenotypic effects resulting from transgene expression. The random insertional mutagenesis is a valuable by-product of the generation of transgenic mice causing a wide variety of phenotypic effects. In approximately 5-10% of transgenic animals, the coding sequence of endogenous genes is disrupted and their expression is suppressed. Furthermore, the inserted DNA tags the mutated genes and enables their cloning and identification. However, tandem arrays of transgenes can also generate chromosomal rearrangements and deletions and thereby hinder the isolation of the affected genes, precluding insertional mutagenesis by transgenes from being developed for the effective knockout of genes with high throughput (Gridley, 1991; Jaenisch, 1988; Meisler, 1992). In strong contrast to the hit-and-miss nature of these highly random mutagenesis strategies is gene targeting, the deletion of single genes with a molecularly well-defined nature in the germ line of mice. Gene targeting was enabled by two principal developments in the 1980s. First, methods were developed to culture ES cells derived from mouse blastocysts in vitro (Evans and Kaufman, 1981; Martin, 1981). Furthermore, after injection into host blastocysts and reimplantation of the embryos into foster mothers, these ES cells were shown to be capable of contributing to all tissues in chimeras, including the germ line (Bradley et al., 1984). Second, it was shown that ES cells can be genetically manipulated and that mutated ES cells are still germ line competent. The method was further perfected by the possibility to introduce directed, precise, and preplanned mutations into any gene in ES cells by the homologous recombination method. Therefore, a cloned targeting vector containing a combination of sequences homologous to the wild-type (WT) gene and the desired mutation is microinjected into ES cells. After homologous recombination, the desired mutation gets part of the mouse genome and replaces the endogenous sequence (Doetschman et al., 1987; Thomas and Capecchi, 1987). For the development of these groundbreaking techniques, Mario R. Capecchi, Martin Evans, and Oliver Smithies were awarded the Nobel Prize for Medicine in 2007 because the combination of gene targeting (Doetschman et al., 1987; Thomas and Capecchi, 1987) with ES cell culture (Evans and Kaufman, 1981; Martin, 1981) led to the generation of the first KO mouse in 1989. An emerging technology for post-genomic mutation of gene expression is RNA interference (RNAi), which is mediated by small interfering RNA (siRNA). siRNA like miRNA is a class of double-stranded RNA molecules comprising 20-25 bp. siRNA binds to complementary sequences on target mRNA transcripts, usually resulting in translational repression or target degradation and gene silencing. To induce long-term knockdown phenotypes in mice, stable siRNA expression constructs have been developed. They integrate into the mouse genome
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3. Introduction after microinjection of DNA into one cell mouse embryos or after transfecting ES cells. These vectors contain RNA polymerase III promoter-driven expression elements of short hairpin RNA (shRNA), which is further processed to the silencing siRNA (Hasuwa et al., 2002; Kunath et al., 2003). However, RNAi-mediated knockdowns often show some persisting residual expression of the silenced gene and the knockdown efficiency is variable in different cell types. Furthermore, siRNA gene knockdown may also exert non-specific effects, e.g. initiate an immune response because it may be recognized as a viral by-product within the cell, or inadvertently downregulate genes with incomplete complementarity.
3.3.2. Gene Trapping ‐ Knockout on the Fast Lane
In comparison to completely random mutations induced by radiation, chemical mutagens, or retroviral/transgene insertion, and in contrast to molecularly defined mutations generated by gene targeting, gene trapping takes a middle path by combining random insertional mutagenesis with the ability to easily identify and clone the mutated gene. For this mutagenesis strategy, a gene trap vector is introduced into the mouse genome by electroporation or retroviral infection of ES cells, ES cells with the integrated gene trap vector are injected into blastocysts, and the latter are reimplanted into foster mothers (Figure 6). A standard gene trap vector consists of a promoterless reporter gene, e.g. lacZ, which allows expression profiling of endogenous promoter activity. It further comprises a selectable genetic marker, mostly an antibiotic resistance gene like neoR, which enables the positive selection of ES cells with gene trap vector integration in antibiotic-containing culture medium. These genes are flanked upstream by a splice acceptor site and intronic sequences, and downstream by a polyadenylation sequence and plasmid vector backbone, ensuring a proper vector integration, splicing, transcription, and translation (Figure 12). Thus, a gene trap vector also provides a DNA tag for the rapid identification of the disrupted gene (Ullrich and Schuh, 2009). Gene trap vectors are designed to function when inserted in an intron of an expressed gene. After intronic insertion, the gene trap cassette is transcribed from the promoter of the trapped gene, resulting in the expression of a fusion transcript, in which the exon(s) upstream of the insertion site is spliced in frame to the reporter/selectable marker gene. Since transcription is terminated prematurely at the inserted polyadenylation site, the processed fusion transcript encodes a truncated or disrupted non-functional version of the affected endogenous gene and the reporter/selectable marker gene. Thus, gene trapping is a powerful tool to simultaneously ablate gene function and to report expression and promoter activity of the trapped gene (Ullrich and Schuh, 2009).
27
3. Introduction
Figure 6: General Gene Trapping Strategy By electroporation of a gene trap vector into murine ES cells, a gene is disrupted randomly by integration of the gene trap vector into the ES cell genome. ES cells containing a random modification in the desired gene are selected, microinjected into fertilized and isolated mouse blastocysts, and re-implanted into the uterus of pseudo-pregnant foster mothers. Chimeric male offspring born from these embryos are mated with female WTs in order to transmit the modified gene to their progeny. Selected heterozygous (HET) female and male mice are mated with each other to generate complete KO mice. Figure in part reproduced from: http://proj.ncku.edu.tw/research/commentary/e/20071116/images/c2_1.jpg.
Despite of less flexibility in the design of the mutated alleles, despite the preference of gene trap vectors to integrate to large transcription units and genes highly expressed in ES cells, and despite the uncertainty to generate complete null alleles, gene trapping has striking advantages compared to gene targeting. Besides the possibility to mutate and report the expression of the trapped gene in parallel, gene trapping is rapid, cost-effective, and does not require the knowledge of structure and sequence of the mutated gene. Accordingly, the long-lasting time- and work-consuming cloning of homologous recombination vectors essential for gene targeting is not necessary. Due to the requirement of only one common and standardized gene trap vector for all knockout approaches performed by gene trapping, this method enables the fast and easy generation of a large variety of insertional mutations throughout the whole mouse genome and therefore is amenable to high throughput and large-scale mutagenesis.
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3. Introduction
The International Gene Trap Consortium (IGTC) consists of laboratories all around the world working together to generate a public library of all mutated murine ES cell lines. The IGTC ensures a broad availability of a wide collection of ES cell clones with a huge variety of identified trapped genes that can be obtained on a non-collaborative basis for nominal handling fees by every scientist. In addition to ES cell clones producing standard loss-of-function alleles, the IGTC also offers ES cell lines with newer gene trap vectors that enable post-insertional modification for the generation of other experimental alleles. In these new vectors, Cre/loxP (cyclisation recombination/locus of X-over of P1) and/or FLP/FRT (flippase/flippase recognition target) recombinase systems are combined with conventional gene trapping strategies. This allows, for example, the rescue and conversion of complete KOs that result in embryonic lethality to temporally and spatially-controllable conditional KOs in a single mouse line. The IGTC, together with the members of the International Mouse Phenotyping Consortium (IMPC), aim to mutate all protein-coding genes in the mouse, using a combination of conventional/conditional gene trapping and gene targeting in C57BL/6 mouse ES cells. Owing to the continuous progress of these large-scale mouse mutagenesis programs, targeted and/or trapped ES cells are available for about 18,000 of the approximately 25,000 identified protein- coding mouse genes to date.
3.4. Hormonal Regulation of Body Homeostasis
3.4.1. The Hypothalamic‐Pituitary‐Adrenal Axis
The hypothalamic-pituitary-adrenal (HPA) axis is essential for the maintenance of homeostasis and enables the organism to prepare for and manage either physical or emotional stress. It comprises a complex set of direct influences and feedback interactions among the hypothalamus, the pituitary gland, and the adrenal glands and constitutes a major part of the neuroendocrine system. Triggered by the limbic system, activation of the HPA axis begins with the release of corticotropin-releasing hormone (CRH) from the hypothalamus, which in turn stimulates the secretion of adrenocorticotropic hormone (ACTH) from the anterior pituitary and further results in the release of mineralocorticoids like aldosterone and glucocorticoids like cortisol or corticosterone from the adrenal gland (Figure 7).
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Figure 7: The HPA Axis – A Crucial Hormonal Mediator of Stress Response and Body Homeostasis Triggered by the limbic system, CRH is released from the PVN of the hypothalamus, which in turn stimulates the secretion of ACTH from anterior pituitary. ACTH mainly triggers the release of glucocorticoids like cortisol or corticosterone from the zona fasciculata of the adrenal cortex. ACTH is also involved in the release of mineralocorticoids like aldosterone from the adrenal zona glomerulosa and of androgens from the adrenal zona reticularis and further influences the secretion of catecholamines from the adrenal medulla. AVP is mainly secreted by the posterior pituitary. Negative feedback regulation is exerted by glucocorticoids, which can inhibit both ACTH and CRH release and directly the limbic system. ACTH can further feed back on CRH secretion. Figure in part reprinted from http://histoblog.viabloga.com/images/hypophyse_1_t.800.jpg and from http://www.gru.edu/mcg/phy/raineylab/images/webfigure2.jpg.
3.4.1.1. The Hypothalamus – Neural Stimuli Trigger the Release of CRH and AVP
The hypothalamus is a part of the brain located between thalamus and brain stem and contains a number of small nuclei with a variety of functions. It links the nervous system to the endocrine system by the secretion of hypothalamic releasing hormones, e.g. corticotropin-releasing hormone (CRH), thyrotropin-releasing hormone (TRH), gonadotropin-releasing hormone 30
3. Introduction
(GnRH), and growth hormone-releasing hormone (GHRH) and by secretion of release-inhibiting hormones, e.g. somatostatin and dopamine. Triggered by the autonomic nervous system, the release of these liberins and statins controls body temperature, hunger, thirst, fatigue, sleep, and circadian cycles (McCann and Porter, 1969). The hypothalamic nucleus responsible for the release of CRH is the paraventricular nucleus (PVN). CRH neurosecretory cells are parvocellular neurons within this PVN that project to the median eminence, which connects the hypothalamus with the pituitary gland (Alonso et al., 1986). From there, CRH and other releasing hormones are secreted into the pituitary portal circulation, diffuse to the anterior pituitary, and in turn stimulate or inhibit the secretion of pituitary hormones, e.g. ACTH (Rivier et al., 1982; Zimmerman et al., 1977). CRH, a 41-amino acid peptide that is cleaved from the C-terminus of a 196-amino-acid pre- prohormone precursor by PC1 and PC2 (prohormone convertases 1 and 2), is the main activator of the HPA axis (Vale et al., 1981). Stress causes rapid activation of CRH neurons by activation of neural pathways afferent to the PVN, resulting in rapid CRH release followed by increases in CRH transcription and de novo synthesis of the peptide. Systemic physical and metabolic stressors, such as loss of blood volume, immune challenge, pain, and hypoglycemia, require an immediate response. They utilize monosynaptic ascending pathways from the brain stem and spinal cord with direct projections to the PVN (Aguilera and Liu, 2012; Herman et al., 2003). These direct pathways to the CRH neuron are mostly noradrenergic and adrenergic and stimulate CRH neurons by acting via α-adrenergic receptors in the CRH neuron. Psychogenic stressors utilize complex polysynaptic pathways, projecting from sensory organs and brain stem to limbic structures, the most important being the medial prefrontal cortex, hippocampus, and amygdala (Aguilera and Liu, 2012; Herman et al., 2003). These pathways interact with the CRH neuron indirectly through glutamatergic and GABAergic neurons (GABA=γ-aminobutyric acid) in the peri-PVN area, which form direct synapses with the CRH neuron. Under basal conditions, the CRH neuron is under inhibitory influence of GABAergic interneurons located in the periventricular region of the hypothalamus (Cullinan et al., 2008). Acute stress induces Ca2+-dependent rapid release of CRH, which is followed by rapid increases in gene transcription in order to restore releasable pools, resulting in an elevation of steady-state mRNA levels detectable about 2 h after acute stress (Ma et al., 1997). During chronic stress, changes in CRH expression in parvicellular neurons depend on the nature of the stress. Whereas psychogenic stressors induce transient increases in mRNA levels, returning to basal after the first few exposures to the stimulus, systemic paradigms cause elevations in mRNA levels up to 14 or more days after each stress exposure (Aguilera, 1998). AVP, a nine-amino acid peptide derived from a preprohormone precursor, is not essential for activation of the HPA axis and has only a weak effect on ACTH release. However, AVP clearly
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3. Introduction potentiates the stimulatory effect of CRH on ACTH release in the pituitary corticotrope cells (Gillies et al., 1982; Rivier and Vale, 1983). Like CRH, AVP responsible for ACTH secretion is produced in the parvocellular neurons of the PVN and released into pituitary portal capillaries from axon terminals of the median eminence (Sawchenko et al., 1984; Zimmerman et al., 1977). In analogy to CRH, the synergistic hypothalamic AVP release is stress-stimulated and also involves an increase in AVP transcription (Aguilera et al., 2008; Ma et al., 1997). In order to exert it effects, CRH binds to CRH receptors 1 and 2 (CRH-R1/2) (Chen et al., 1993;
Lovenberg et al., 1995), which are mainly coupled to Gs proteins (Grammatopoulos and Chrousos, 2002). Whereas CRH-R1 has been implicated in mediating normal responses to stress, CRH-R2 seems to be more responsible for fine tuning and counterregulation (Timpl et al., 1998). For stress-induced activation of the HPA axis and the initiation of biosynthesis and secretion of ACTH from cellular stores, CRH acts exclusively on CRH-R1 receptors. The synergistic action of AVP on ACTH release is mediated by Gq protein-coupled vasopressin 1b receptors (V1bR) in the anterior pituitary (Birnbaumer, 2000; Sugimoto et al., 1994). Both the resulting increase in cyclic adenosine monophosphate (cAMP) and intracellular Ca2+ lead to a stimulation of pro-opiomelanocortin (POMC) transcription and ACTH production (Jenks, 2009).
3.4.1.2. The Pituitary – CRH/AVP‐mediated ACTH Release from Anterior Lobe and Osmolality/Volume‐mediated AVP Release from Posterior Lobe
The pituitary is an endocrine gland at the base of the brain, resting in a small, bony cavity and covered by a dural fold. It is not a part of the brain but a protrusion of the hypothalamus with whom it is functionally connected by the median eminence. The pituitary gland consists of two components: the anterior pituitary or adenohypophysis and the posterior pituitary or neurohypophysis. Regulated by the hypothalamic liberins and statins, the anterior pituitary synthesizes and secretes tropic hormones that target other endocrine glands to release further downstream hormones, e.g. adrenocorticotropic hormone (ACTH or corticotropin), growth hormone (GH), thyroid-stimulating hormone (TSH or thyrotropin), luteinizing hormone (LH), follicle-stimulating hormone (FSH), prolactin (PRL), and melanocyte-stimulating hormone (MSH) (McCann et al., 1968). The posterior pituitary stores and secretes AVP, the majority of which is produced in the supraoptic nucleus (SON) of the hypothalamus and oxytocin, most of which is generated in the hypothalamic PVN (Hayward, 1975). In contrast to the parvocellular CRH neurons in the hypothalamic PVN, which also produce and release small amounts of AVP (Sawchenko et al., 1984; Zimmerman et al., 1977), the main source of AVP are magnocellular neurons of the hypothalamic SON and PVN, which are also responsible for oxytocin release (Taniguchi et al., 1988). AVP is transported to the posterior 32
3. Introduction pituitary along these magnocellular neurons, where it is stored in vesicles and from where it is released to the peripheral circulation. AVP of magnocellular origin exerts the major AVP- hormone function, i.e. the regulation of body fluid homeostasis by mediating water retention in the kidney. Osmotic stressors like hyperosmolality or hypovolemia activate vasopressinergic neurons leading to opening of voltage-gated Ca2+ channels in these nerve terminals, transient Ca2+ influx, and the fusion of the AVP-containing neurosecretory granules with the nerve terminal membrane (Benetos et al., 1987; Dunn et al., 1973; Ishikawa and Schrier, 1983). AVP exerts its antidiuretic, water-retaining effect by binding to V2Rs, expressed in the kidney in cells of the distal convoluted tubule and in principle cells of the collecting duct (Lolait et al.,
1992). V2Rs in the basolateral membrane of these cells are associated with Gs proteins, which trigger the insertion of aquaporin-2 (AQP2) water channels into the apical cell membranes, involving cAMP increase and protein kinase A (PKA) activation. Aquaporins allow water to follow its osmotic gradient, leading to water re-absorption from the nephron back into the circulation (Nielsen et al., 1996). Besides the regulation of body fluid homeostasis, circulating AVP at high concentrations also elicits vasoconstriction and raises blood pressure. This effect is 2+ mediated by V1aR and Ca , similar to V1bR a Gq protein-coupled receptor (Koshimizu et al., 2012; Morel et al., 1992). In the anterior pituitary, the neurohormones CRH and AVP act synergistically via their specific CRH-R1 and V1bRs, respectively, to trigger the release of ACTH from corticotrope cells into the systemic circulation (Sawchenko et al., 1984; Zimmerman et al., 1977). ACTH is a 39-amino acid peptide but is synthesized as part of the 241-amino acid polypeptide POMC. POMC is synthesized primarily in the CNS by corticotrope cells of the anterior pituitary, by melanotrope cells of the intermediate lobe of the pituitary, and in the arcuate nucleus of the hypothalamus. It is also present in several peripheral tissues, e.g. in melanocytes in the skin, in adrenals, lung, spleen, in genitourinary- and gastrointestinal tracts, and in thyroid and immune cells (Eberle, 1988; Wikberg et al., 2000). The polypeptide hormone precursor POMC undergoes extensive, tissue-specific, post-translational processing via cleavage by endopeptidases, mainly PC1 and PC2. Further processing by phosphorylation, acetylation, amidation, and glycosylation contributes to the production of various polypeptide fragments by different cell types with varying physiological activity (Smith and Funder, 1988). In the anterior pituitary, POMC is cleaved to γ-melanocyte-stimulating hormone (γ-MSH), ACTH, and β-lipotropin (β-LPH). ACTH can further be processed to α-MSH and corticotropin-like intermediate lobe peptide (CLIP), whereas β-LPH can further be cleaved to γ-LPH and β-endorphin. From γ-LPH, even more β- MSH can be generated. Each of these peptides is packaged in large dense-core vesicles that are released by exocytosis in response to CRH/AVP-mediated stimulation (Smith and Funder, 1988).
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ACTH and the other POMC cleavage products exert their numerous biological effects by activating melanocortin receptors, a family of five Gs-protein coupled receptors (MC1-5R) (Mountjoy et al., 1992). The MCRs are mainly expressed in melanocytes, endothelial cells, fibroblasts, immune cells, CNS, and brain and therefore play important functional roles in the regulation of behavior, skin pigmentation, energy homeostasis, feeding, immune system, cardiovascular system, and sexual function. The main ligands of the MCRs are the melanocortins, especially α-MSH, which has the highest receptor affinity in most cases. ACTH is able to bind to all five MCRs but in most cases with a profoundly lower affinity in comparison to the melanocortins. One exception is MC2R, which is exclusively responsive to ACTH and not to the MSHs (Gantz and Fong, 2003; Getting, 2006). MC2R is located in the adrenal zona glomerulosa and zona fasciculata, where it regulates steroidogenesis upon ACTH binding (Liakos et al., 1998).
3.4.1.3. The Adrenal – ACTH Regulates the Release of Glucocorticoids and Mineralocorticoids
The adrenal glands are endocrine glands sitting at the top of the kidneys. Each adrenal gland has two distinct areas, the outer adrenal cortex and the inner medulla (Figure 7). The adrenal cortex comprises three zones that differ anatomically and functionally: an outer zona glomerulosa, a central zona fasciculata, and an inner zona reticularis. Whereas the zona glomerulosa is the major site for production of mineralocorticoids, mainly aldosterone, which is largely responsible for regulation of salt and water homeostasis and blood pressure, the zona reticularis produces androgens, mainly dehydroepiandrosterone (DHEA) and androstenedione, the precursor of testosterone and estradiol in humans. Both the zona fasciculata and the medulla respond to stress, the former by the synthesis of corticosteroids such as cortisol and the latter by the release of catecholamines such as epinephrine or norepinephrine. Whereas the medullary release of catecholamines developed from tyrosine is triggered mainly and directly by the sympathetic nervous system, the release of cortical hormones developed from cholesterol is regulated by hormones itself, of which ACTH plays a major role. Human steroidogenesis of mineralocorticoids, glucocorticoids, and androgens starts with the conversion of the common precursor cholesterol to pregnenolone by cholesterol side chain cleavage monooxygenase, also called P450scc and encoded by the CYP11A1 gene (Figure 8). For glucocorticoid synthesis, which takes place in the adrenal zona fasciculata, pregnenolone is converted to 17α-hydroxypregnenolone by 17α-hydroxylase (P450c17/CYP17A1). Consecutive catalytic activity of 3β-hydroxysteroid dehydrogenase (3β-HSD/HSD3B), 21α-hydroxylase (P450c21/CYP21A2), and 11β-hydroxylase (P450c11/CYP11B1) leads via 17α- 34
3. Introduction hydroxyprogesterone and 11-deoxycortisol to the generation of the glucocorticoid cortisol. Mineralocorticoid synthesis takes place accordingly in the zona glomerulosa using almost the same steroidogenic enzymes with the exception of 17α-hydroxylase and 11β-hydroxylase, which are not expressed there. Via pregnenolone and progesterone, cholesterol is converted to 11-deoxycorticosterone, mediated by consecutive action of P450scc, 3 β-HSD and P450c21. The last step in aldosterone synthesis is mediated by aldosterone synthase (P450aldo/CYP11B2), which catalyzes the 11β-hydroxylation to corticosterone, the 18- hydroxylation to 18-hydroxy-corticosterone, and the 18-oxidation to aldosterone. The generation of adrenal androgens is also catalyzed by 17α-hydroxylase, which converts 17α- hydroxypregnenolone to dehydroepiandrosterone and 17α-hydroxyprogesterone to androstenedione. These steroids are precursors of testosterone and estradiol (for review of steroidogenesis see (Hattangady et al., 2012; Sewer and Waterman, 2003)).
Figure 8: Adrenal Synthesis of Human Cortisol and Aldosterone Mineralocorticoids in the adrenal zona glomerulosa and glucocorticoids in the zona fasciculata are generated from their common precursor cholesterol mediated by partially identical steroidogenic enzymes. The reactions catalyzed by CYP11A1 (cholesterol side chain cleavage monooxygenase), CYP11B1 (11β-hydroxylase), and CYP11B2 (aldosterone synthase) occur in the mitochondria, all other reactions take place in the endoplasmic reticulum. Enzymes specifically present in the zona fasciculata are CYP17A1 (17α-hydroxylase) and CYP11B1, while CYP11B2 is exclusively expressed in the zona glomerulosa. Figure based on (Morohashi et al., 2013).
In contrast to the human steroidogenesis described above, murine adrenal glands are not able to produce cortisol and androgens due to the lack of CYP17A1 expression (Figure 9). Therefore, in mice corticosterone and not cortisol is the predominant steroid of the adrenal cortex (Keegan and Hammer, 2002). 11-deoxycorticosterone is the common precursor for the production both aldosterone in the zona glomerulosa and corticosterone in the zona fasciculata. After binding to MC2R, ACTH regulates steroidogenesis by adenylate cyclase stimulation and PKA activation (Penhoat et al., 1989). Acutely, PKA phosphorylates steroidogenic acute regulatory protein (StAR), which mediates the delivery of the initial substrate cholesterol from
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3. Introduction cellular stores to mitochondrial P450scc and therefore leads to a rapid cholesterol mobilization (Clark et al., 2000). Chronically, ACTH-dependent PKA activation stimulates the transcription of all steroidogenic CYP enzymes (CYP11A1, CYP17, CYP21A2, CYP11B1) with the exception of CYP11B2 (Holland and Carr, 1993; Sewer and Waterman, 2002, 2003). Because ACTH is able to stimulate especially 11β-hydroxylase transcription but has no influence on aldosterone synthase expression, ACTH has a greater impact on glucocorticoid than on mineralocorticoid regulation (Kramer et al., 1983).
Figure 9: Synthesis of Corticosterone and Aldosterone in Mice In contrast to humans, rodents are not able to synthesize cortisol and steroidogenesis of mineralocorticoids and glucocorticoids is equal up to 11-deoxycorticosterone. In the zona fasciculata, 11- deoxycorticosterone is converted exclusively to corticosterone by 11β- hydroxylase. In the zona glomerulosa, 11-deoxycorticosterone is also converted to corticosterone, in this zone mediated by aldosterone synthase, which is exclusively expressed there and which in a further two-step reaction generates aldosterone from corticosterone. Figure based on http://upload.wikimedia.org/wikipedia/commons/8/89/ Corticosteroid-biosynthetic-pathway-rat.png.
Glucocorticoids are a class of steroid hormones that are released in response to stress. Both cortisol and corticosterone respond to psychological but also physiological stress by enabling the mobilization of energy reserves. They exert effects on glucose, protein, and fat metabolism. Glucocorticoids stimulate gluconeogenesis by simultaneous inhibition of enteral glucose reabsorption and glucose uptake in muscles and adipose tissue, by proteolysis and mobilization of amino acids from peripheral tissues, and by lipolysis and the release of fatty acids, the latter two mechanisms providing substrates for gluconeogenesis. They have an immune-suppressive function by upregulating the expression of anti-inflammatory proteins and downregulating the expression of pro-inflammatory proteins. Together with catecholamines, glucocorticoids enhance cardiac power and blood pressure and cooperate in memory formation, e.g. of events associated with strong emotions, both positive and negative.
Whereas both CRH-R and MC2-R are membrane-bound Gs-protein-coupled receptors linked to the adenylate cyclase - cAMP - PKA pathway, glucocorticoid receptors are cytosolic steroid receptors. In the absence of hormones, the glucocorticoid receptor resides in the cytosol and forms a complex with a variety of proteins including heat shock proteins (Pratt et al., 2006). The
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3. Introduction glucocorticoid hormone cortisol diffuses through the cell membrane into the cytoplasm and binds to the glucocorticoid receptor (GR) resulting in release of the heat shock proteins. The resulting activated GR complex has two principal mechanisms of action: Transactivation involves receptor homodimerization upon ligand binding, translocation into the cell nucleus, and interaction with specific glucocorticoid response elements (GRE) in the promoter region of the target genes, thus activating gene transcription (Mangelsdorf et al., 1995). Via transrepression, activated glucocorticoid receptors can complex with other transcription factors, such as NF-κB (Ray and Prefontaine, 1994) or AP-1 (Jonat et al., 1990), prevent them from binding their target genes, and repress gene expression normally upregulated by these factors.
3.4.1.4. Feedback Regulation and Circadian Rhythm of the HPA Axis
The activity of the HPA axis is determined by endogenous inputs clocked by the circadian rhythm and by exogenous inputs elicited by stress, both mediated by neural modulation and by glucocorticoid feedback control. Glucocorticoids exert a negative feedback on the HPA axis at the level of pituitary, hypothalamus, and centrally at the level of the limbic system (Figure 7). Feedback inhibition is mediated via the GR and involves fast and slow feedback loops (Dallman et al., 1994; Keller- Wood and Dallman, 1984; Papadimitriou and Priftis, 2009). Slow feedback is mediated by glucocorticoid binding to cytosolic GRs and involves genomic regulation. In this manner, glucocorticoid production by HPA axis activation leads to a consecutive downregulation of CRH and AVP mRNA synthesis in the hypothalamus and of POMC gene transcription in the anterior pituitary (Birnberg et al., 1983; Kovacs et al., 2000; Kretz et al., 1999). Fast feedback regulation of the HPA axis occurs by acute inhibition of CRH and ACTH release. The rapidity of action from seconds to minutes suggests that this effect is not compatible with genomic regulation. Furthermore, rapid effects seem to be mediated by binding of glucocorticoids to membrane- associated glucocorticoid receptors instead of to the classical cytosolic ones (Tasker et al., 2006). Glucocorticoid binding to these membrane-bound receptors leads to the release of endocannabinoids, which exert acute, fast feedback by inhibition of glutamatergic stimulation of the PVN neurons (Di et al., 2003). In the absence of negative feedback control of ACTH secretion by glucocorticoids, ACTH can regulate its secretion by inhibition of hypothalamic CRH release, which also represents a fast feedback loop (Suda et al., 1987) (Figure 7). Furthermore, the release of the HPA hormones underlies a circadian rhythm. This circadian rhythmicity is generated by the hypothalamic suprachiasmatic nucleus (SCN) dependent on the sleep-wake cycle of the organism (Ralph et al., 1990; Rusak and Zucker, 1979). The SCN triggers the rhythmic release of CRH, which in turn mediates the secretion of ACTH in a
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3. Introduction pulsatile fashion. Under basal conditions, approximately 18 - 25 pulses of ACTH with various amplitudes are observed over 24 h. Depending on the light-dark cycle, maximum levels of ACTH are reached at a 2 - 4 h window around waking and subsequently decrease to a minimum at a 2 - 4 h window around sleeping. Corticosterone concentration profiles follow those of ACTH with approximately 2 h delay (Oster et al., 2006). Dysregulation of the HPA axis, both up- or downregulation, plays a fundamental role in various stress-associated and psychiatric (Stokes and Sikes, 1991), neurodegenerative (Bao et al., 2008), metabolic (Williams, 1979), and chronic pain and inflammatory diseases (Bomholt et al., 2004). Increased activity of the HPA axis is associated with mood disorders, including e.g. obsessive-compulsive disorder, anxiety disorder, eat disorders, insomnia, major depressive disorder, and alcoholism (Arborelius et al., 1999; Kluge et al., 2007; Pariante, 2003; Stokes and Sikes, 1991). A metabolic consequence of HPA axis overactivation is hypercortisolism and the thereof resulting Cushing´s syndrome (Williams, 1979). The most common consequence of decreased HPA activity is adrenal insufficiency, also called Addison´s disease, and hypocortisolism (Williams, 1979), but it can also be associated with mood disorders like chronic fatigue syndrome (Papadopoulos and Cleare, 2012).
3.4.2. The Renin‐Angiotensin‐Aldosterone Axis
The Renin-Angiotensin-aldosterone-axis is a major regulator of extracellular fluid and salt balance and therefore the most important cascade for blood pressure regulation. It comprises a well-characterized cascade of proteases and hormones, of which the sequentially activated peptidase Renin and the octapeptide Angotensin II are the most important and physiologically active components. Angotensin II acts on the adrenal gland to release the mineralocorticoid aldosterone (Figure 10).
3.4.2.1. The Renin‐Angiotensin System (RAS)
Renin is the initiator of the RAS and is derived from a Prorenin precursor by proteolytic cleavage (Galen et al., 1984). It is synthesized and stored in juxtaglomerular cells of the kidney, a specialized form of granulated smooth muscle cells located in the wall of the afferent arteriole of a nephron (Tobian, 1962). The peptidase is released from the juxtaglomerular apparatus into the blood circulation and generates Angiotensin I (Ang I) from its substrate angiotensinogen by proteolytic cleavage. Angiotensinogen is mainly produced by the liver (Nasjetti and Masson, 1971) and contains more than 400 amino acids. However, only the first ten amino acids represent Ang I (Tewksbury et al., 1981). The decapeptide Ang I is biologically mostly inactive 38
3. Introduction and serves as a precursor for the physiologically active Ang II. Ang II, an octapeptide, contains the first eight amino acids of Ang I and is generated by angiotensinogen-converting-enzyme (ACE) (Ng and Vane, 1967). The exopeptidase ACE is secreted by pulmonary and renal endothelial cells and removes the two C-terminal residues of Ang I (for reviews of the RAS see (Bottari et al., 1993; Fyhrquist and Saijonmaa, 2008)).
Figure 10: The Renin-Angiotensin System – The Main Stimulator of Aldosterone Release Renin, derived from its precursor Prorenin, is secreted by juxtaglomerular cells in the kidney and generates Ang I from angiotensinogen by proteolytic cleavage. By Angiotensin converting enzyme, Ang I is further processed to Ang II, which stimulates the release of aldosterone from the adrenal cortex. Ang II can directly inhibit the RAS by suppressing renal Renin release, while aldosterone indirectly signals back to Renin release by aldosterone-induced volume expansion. Figure in part reproduced from http://www.paradisi.de/images_artikel/1/12556_0.jpg and from http://www.gru.edu/mcg/phy/raineylab/images/webfigure2.jpg.
The secretion of Renin and the initiation of the RAS is mainly provoked by three situations: First, by a decrease in arterial blood pressure and a decrease in blood volume, respectively, which is detected by renal baroreceptors (Blaine and Davis, 1971). Second, by the sympathetic nervous system, which innervates juxtaglomerular cells and activates the RAS in case of hypotension by acting through adrenaline-binding to β1-adrenergic receptors (Gordon et al., 1967; Vander, 1965). Third, by a decrease in the glomerular filtration rate (GFR) and by thereof dependent reduced Na+ levels in the ultra-filtrate of the nephron. The NaCl content and the renal blood flow is sensed indirectly by a Na+ K+ 2Cl- symporter, which measures the speed of ion transport in the macula densa, a collection of densely packed epithelial cells in the distal tubule of the nephron (Skott and Briggs, 1987). The RAS has a number of physiological effects, all contributing to blood pressure and fluid-salt balance regulation. Ang II, the main effector of the RAS, is a potent vasoconstrictor of arterioles throughout the whole body and thus has the potential to raise systemic arterial blood pressure (Basso and Terragno, 2001). In the kidney, Ang II maintains renal blood flow and GFR independently of arterial blood pressure changes. In this manner, Ang II provokes vasoconstriction of renal efferent arterioles in case of hypotension and of afferent arterioles in case of hypertension (Davalos et al., 1978; Heyeraas and Aukland, 1987). In the CNS, Ang II increases thirst sensation and the desire for salt (Avrith and Fitzsimons, 1978; Epstein et al., 39
3. Introduction
1969). Accordingly, it influences salt and water homeostasis by supporting the release of AVP from the posterior pituitary and by acting on the adrenal cortex to release aldosterone (Carpenter et al., 1961; Shimizu et al., 1973). Ang II exerts its effects via two Ang II receptors mediating different physiological effects and exerting different mechanisms of action (Gunther, 1984). The Ang II receptor type 1 (AT1), mainly Gq- and sometimes Gi-protein-coupled, is involved in most of the well-established physiological effects of Ang II. It regulates vasoconstriction, aldosterone and ADH secretion but also cell proliferation and cellular growth, generation of reactive oxygen species, and inflammation (Fyhrquist and Saijonmaa, 2008). Thus, the AT1 receptor also mediates actions with potentially harmful consequences, i.e. hypertension, cardiac hypertrophy, and fibrosis. The
Gi-protein-coupled Ang II receptor type 2 (AT2) was shown to counterbalance some effects of the AT1 receptor by mediating vasodilatation and inhibition of proliferation and growth (de Gasparo et al., 2000; Kaschina and Unger, 2003).
3.4.2.2. Aldosterone
Aldosterone, like corticosterone and cortisol a steroid hormone, is the primary human mineralocorticoid and is produced in the zona glomerulosa of the adrenal gland cortex (Giroud et al., 1956). Steroidogenesis of aldosterone starts with cholesterol and is carried out by nearly the same enzymes required for steroidogenesis of cortisol (3.4.1.3., Figure 8) (Hattangady et al., 2012). In contrast to cortisol biosynthesis, aldosterone biosynthesis lacks 17-hydroxylation of pregnenolone and progesterone, respectively, because the zona glomerulosa is not able to express P450c17 (Narasaka et al., 2001). In mice, where corticosterone is the main glucocorticoid, sterodogenesis of mineralocorticoids and glucocorticoids is equal up to 11- deoxycorticosterone (Figure 9). The last steps from 11-deoxycorticosterone to aldosterone are mediated by aldosterone synthase, which is exclusively expressed in the zona glomerulosa (Domalik et al., 1991; Ogishima et al., 1992). This zone-specific expression of P450aldo and the lack of P450c17 and P450c11, which are required for glucocorticoid synthesis, help to maintain the functional specificity of the adrenocortical zones and restrict aldosterone production to the zona glomerulosa. Aldosterone biosynthesis is, similar to that of cortisol, regulated acutely by the StAR-mediated movement of cholesterol to P450scc in the mitochondria and chronically by aldosterone synthase expression. The main initiators of aldosterone release are Ang II, K+, and ACTH. For acute regulation, all three stimulators mainly exert their effects by increasing cytosolic Ca2+ concentrations, by stimulating Ca2+/calmodulin-dependent protein kinases (CaMK) or PKA, and at last by activating CREB (cAMP responsive element binding protein) and StAR (Cherradi et
40
3. Introduction al., 1998; Clem et al., 2005; Manna et al., 2003; Pezzi et al., 1996; Stocco, 2001). Chronic regulation is mainly exerted by the expression of aldosterone synthase, which is primarily induced by Ang II- and K+-stimulated increase in intracellular Ca2+ and activation of CaMK (Condon et al., 2002; Holland and Carr, 1993; Tremblay et al., 1992).. Aldosterone synthase expression is regulated directly by transcription factors like NURR1 (nuclear receptor related 1), NGF1B (nerve growth factor 1 B), ATF1 (cyclic AMP-dependent transcription factor), and CREB, which can be activated and phosphorylated by CaMKs (Bassett et al., 2004; Matthews et al., 1994). In contrast to chronic Ang II and K+ effects, chronic ACTH release, however, rather decreases aldosterone secretion by decreasing CYP11B2 expression (Holland and Carr, 1993). The effects of aldosterone are mediated by the mineralocorticoid receptor (MR), whose structure and mode of action is very similar to the GR (3.4.1.3.) (Arriza et al., 1987). Because of its molecular similarity to aldosterone, glucocorticoids can also bind to the mineralocorticoid receptor with almost equal affinity (Arriza et al., 1987; Beaumont and Fanestil, 1983). Although glucocorticoids circulate at much higher concentrations than mineralocorticoids, a specific mineralocorticoid effect is generated by 11β-hydroxysteroid dehydrogenase type II (HSD11B2), existing in mineralocorticoid target tissues, e.g. epithelial cells in kidney and colon or vascular cells. It catalyzes the deactivation of glucocorticoids, i.e. the conversion of cortisol to the inactive metabolite cortisone in man or of corticosterone to 11-dehydrocorticosterone in rat, in order to prevent overstimulation by glucocorticoids and to allow selective mineralocorticoid action (Funder et al., 1988).
Figure 11: Aldosterone and the Regulation of Salt and Water Balance By binding to MRs within distal convoluted tubule cells, connecting tubule cells, and principal cells of the collecting duct, aldosterone modulates directly the transcription of ENaC, ROMK, and SGK1 in the kidney nephron. SGK1 in turn indirectly stimulates the activity of the apical ENaC and ROMK but also of the basolateral Na+-K+-ATPase. Because the latter exchanges 3 Na+ from the tubular cell into the blood and 2 K+ from the blood into the tubular cell, a Na+ gradient develops. This gradient consequently induces the entry of Na+ into the tubular cell at the apical membrane through ENaC, while K+ diffuses into the tubular lumen through apical ROMK. This aldosterone-triggered reabsorption of Na+ and subsequent secretion of K+ controls electrolyte and water levels in the entire body. Figure based on (Kiryluk and Isom, 2007). 41
3. Introduction
The main function of aldosterone is the regulation of extracellular fluid and salt balance. Therefore, aldosterone binds to MRs within distal convoluted tubule cells, connecting tubule cells, and principal cells of the collecting duct in the kidney nephron. Activation of MRs modulates the transcription and/or the activity of several target genes, e.g. epithelial sodium channel (ENaC) (Chen et al., 1999b; Kemendy et al., 1992), renal outer medullary potassium channel (ROMK) (Beesley et al., 1998), Na+-K+-ATPase (Jorgensen, 1972), and serum and glucocorticoid-inducible kinase 1 (SGK1) (Bhargava et al., 2001). Aldosterone seems to be able to directly stimulate ENaC, ROMK, and Na+-K+-ATPase activity in the cell membrane of distal convoluted tubule and collecting duct principal cells by upregulating their mRNA levels (Figure 11). Indirectly, aldosterone induces the transcription of SGK1, which in turn can stimulate the activity of these channels (Kiryluk and Isom, 2007). The basolateral Na+-K+-ATPase exchanges 3 Na+ from the cytosol of the tubular cell into the blood and 2 K+ from the blood into the tubular cell, thereby providing a Na+ gradient into the cell. Consequently, at the apical membrane, Na+ follows this gradient and enters the tubular cell through ENaC, whereas K+ diffuses into the tubular lumen. With this mechanism, aldosterone triggers the reabsorption of Na+ and water into the blood and the subsequent secretion of K+ into the urine. The salt and water retention leads to an increase in blood volume and therefore also in blood pressure.
3.4.2.3. RAS Feedback Regulation, Dysregulation, Antagonists, and Associated Diseases
For the maintenance of salt and water homeostasis, both the RAS and the thereof dependent aldosterone synthesis have to be regulated properly. Renin secretion and RAS activity is terminated through negative feedback loops, which can be induced by physiological stimuli like high arterial blood pressure, subsequently increased renal perfusion pressure, hypervolemia, increased Na+ retention, increased K+ load, and by α-adrenergic activation of the sympathetic nervous system (Williams, 2005). A hormonal feedback regulation is triggered by Ang II itself, which inhibits expression and release of Renin from juxtaglomerular cells (Johns et al., 1990; Vander and Geelhoed, 1965). Aldosterone exerts an indirect negative feedback because aldosterone-mediated salt and water retention leads to volume expansion, which also inhibits Renin release from the juxtaglomerular apparatus (Kaufman et al., 1980) (Figure 10). An endogenous antagonist of the RAS and aldosterone is the atrial natriuretic peptide (ANP), a peptide hormone secreted from cardiac myocytes in the atria of the heart in response to hypervolemia and hypernatremia (Anderson et al., 1986; de Bold, 1985). ANP mediates natriuresis and diuresis and thus is able to downregulate plasma volume and blood pressure (Brenner et al., 1990). 42
3. Introduction
Dysregulation of the Renin-Angiotensin-aldosterone axis plays an important role in the pathogenesis of cardiovascular and renal disorders. Hyperaldosteronism, defined as a pathological increase in aldosterone production and release, may be caused by RAS overactivation (secondary hyperaldosteronism) or may develop independently of the RAS, e.g. due to adrenal adenoma (Conn's Syndrome) or due to adrenal hyperplasia (primary hyperaldosteronism). It is characterized by arterial hypertension, hypernatremia, hypokalemia, and metabolic alkalosis. Hypoaldosteronism accompanied by hypotension, hyponatremia, hyperkalemia, and metabolic acidosis can be provoked by adrenal insufficiency, mutations of steroidogenic genes, e.g. of CYP21A2, aldosterone synthase deficiency, and decreased RAS activity (Torpy et al., 1999). Aldosterone/MR signaling as well as Ang II/AT1 receptor signaling can mediate further actions with harmful potential associated with their designated physiological effects. Common consequences due to or accompanied by overactivation of the Renin-Angiotensin-aldosterone axis are hypertension, thrombosis, hypertrophy, fibrosis, endothelial dysfunction, inflammation, and oxidative stress in cardiac, vascular, and renal tissues. Possible underlying molecular mechanisms are e.g. the stimulation of proliferative and hypertrophic signaling pathways, increased production or decreased degradation of extracellular matrix, and increased reactive oxygen species production (Atlas, 2007; Gekle and Grossmann, 2009; Marney and Brown, 2007; Weir and Dzau, 1999). Due to these unwanted effects, also pharmacological antagonists of the Renin-Angiotensin- aldosterone axis have been developed, e.g. ACE inhibitors, AT1 receptor blockers, β-blockers, and aldosterone antagonists. They are used as therapeutic strategy to treat hypertension and associated cardiovascular and renal disorders. The combination of RAS inhibition and downregulation of AT1 receptor and MR activity, achieved by the simultaneous treatment with two or more of the above mentioned pharmacological antagonists, significantly reduces hypertension, cardiovascular and renal comorbidities, and mortality in patients (Atlas, 2007; Gekle and Grossmann, 2009; Marney and Brown, 2007; Weir and Dzau, 1999).
3.5. Aim of Thesis
In order to investigate physiological functions of the SPRED protein family, first detected and described in 2001 (Wakioka et al., 2001), we generated SPRED2 KO mice by a gene trap approach (Bundschu et al., 2005). SPREDs were identified as inhibitors of exclusively Ras/ERK/MAPK signaling (King et al., 2006; King et al., 2005; Miyoshi et al., 2004; Nobuhisa et 43
3. Introduction al., 2004; Nonami et al., 2004; Sivak et al., 2005; Wakioka et al., 2001), a cascade, which regulates cell proliferation and differentiation in almost every cell in almost every species. Thus, the suppressive effect of SPREDs on the Ras/ERK/MAPK pathway is thought to have widespread regulatory functions throughout the whole body. In a first study, phenotypical characterization of SPRED2-deficient mice up to the age of five months demonstrated that loss of SPRED2 causes dwarfism due to consecutively upregulated ERK activity and thus identified SPRED2 as a novel regulator of chondrocyte differentiation and bone growth (Bundschu et al., 2005). Long term observations of SPRED2 KOs revealed an excessive grooming behavior, an abnormal high water uptake, and a reduced survival due to sudden death. Dissections of older mice or cadavers further disclosed clear signs of kidney deterioration. Therefore, I continued phenotypical characterization of SPRED2 KO mice to unravel these effects of SPRED2 deficiency in adult mice and to discover a common reason causing this versatile phenotype. Due to the complex phenotype and the generally bad health state of SPRED2 KOs, we performed a broad analysis of serum parameters. Because this revealed profound salt and water imbalances, we considered a dysregulation of hormonal homeostasis as a possible cause. The accordingly performed systematic analysis of possibly contributing hormone axes showed that the disequilibrium in fluid and electrolyte balance was associated with hyperaldosteronism but did not affect AVP release. The detection of significantly lowered Ang II levels demonstrated that hyperaldosteronism developed independently of RAS activity. However, it was accompanied by increased aldosterone synthase expression. Aldosterone is together with corticosterone secreted by the adrenal gland, and both are under control of pituitary ACTH and of further upstream hypothalamic CRH. Therefore, we also investigated stress hormone release from the HPA axis, which was completely upregulated at all levels. According to current knowledge, HPA hormone actions are mainly mediated by G protein- coupled receptor pathways and steroid receptor-regulated gene expression. To date, only first insights were obtained how RTKs and MAPK pathways may participate in the regulation of the HPA axis-triggered stress hormone release (Blume et al., 2009; Gerrits et al., 2006; Khan and Watts, 2004; Singru et al., 2008). Thus, we aimed to discover a link between SPRED-mediated inhibition of Ras/ERK/MAPK signaling and HPA axis regulation. Indeed, we detected that SPREDs, by suppressing Ras/ERK/MAPK-mediated activation of Ets transcription factors, can downregulate CRH transcription and release. This explained the hyperactivated HPA axis and the increased endogenous stress response in SPRED2 KO mice, both being reflected in the excessive grooming behavior. The hyperaldosteronism is in part a second consequence of enhanced HPA axis activity but mainly triggered by elevated adrenal aldosterone synthase expression. In SPRED2-deficient mice, this hyperaldosteronism elicits
44
3. Introduction hyperosmolality and polydipsia, the latter further contributing to hydronephrosis, kidney atrophy, and renal apotosis. As a consequence of the gene trapping strategy, a truncated SPRED2 representing a functional EVH1 domain was expressed in our SPRED2 KO mice. Because the EVH1-β-geo expression might be reasonable for the severe and complex phenotype of our SPRED2 KOs, the exploration of its function was of special interest for us, too. In fact, our studies indicated a possible dominant negative effect of the fusion protein on the remaining SPRED1 expression and on Ras/ERK/MAPK signaling. Although this study undoubtedly provided very valuable and interesting insights in phenotypical effects of SPRED2 deficiency, two follow-up studies investigating especially SPRED2 function in heart, and brain and a genetic screen to identify SPRED2-deficient humans are already in progress. Hereby, we aim to further enlighten and compare consequences of SPRED2 deficiency in mice and in humans.
45
46
Materials and Methods 4
4.1. Mouse Handling
4.1.1. Generation of SPRED2 KO Mice
The detailed procedure for the generation of SPRED2 KO mice has already been described previously (Bundschu, 2005; Bundschu et al., 2005; Ullrich and Schuh, 2009). In brief, SPRED2 KO mice were generated from the embryonic stem cell line XB228, which was obtained from BayGenomics, nowadays part of the IGTC. XB228 embryonic stem cells originated from 129P2/OlaHsd mice and contained the pGT0 gene trap vector, which inserted between exons 4 and 5 in the Spred2 gene. XB228 cells with the trapped Spred2 were injected into isolated blastocysts of super-ovulated and fertilized C57BL/6 female mice. Pseudo-pregnant foster mothers that have been mated with vasectomized male mice were used for reimplantation of blastocysts. To establish the SPRED2 KO mouse line, chimeric male offspring were mated to female C57BL/6 WT mice in order to test for germline transmission and to obtain first SPRED2 HET mice.
4.1.2. Mouse Raising and Breeding
Mice were housed in standard plastic cages, either individually or in groups of up to four mice, depending on their gender and the number of littermates. They were kept at a daily constant 12 h light and 12 h dark cycle, 25°C, and at a humidity of 50%. Tap water and standard mouse chow were available ad libitum unless stated otherwise. In order to obtain offspring of all genotypes, i.e. SPRED2 WT, KO, and HET mice with equal ages in the same litter, usually one SPRED2 HET male mouse was mated with two SPRED2
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4. Materials and Methods
HET female mice. To minimize possible inbred effects, mice were bred on a mixed 129P2/OlaHsd x C57Bl/6 genetic background.
4.1.3. Genotyping of SPRED2‐deficient Mice
Mouse offspring was genotyped by PCR analysis using the REDExtract-N-AmpTM Tissue PCR Kit from Sigma-Aldrich (Cat. No. XNATR). According to the protocol, genomic DNA was isolated from mouse tail tip biopsies of approximately 0.5 cm of length by incubating biopsies with a mixture of 100 µl Extraction Solution and 25 µl of Tissue Preparation Solution. Tail tips within the solution were incubated for 12 min at 55°C and boiled for 3 min at 95°C. After addition of 100 µl Neutralization Solution B, tissue extracts were ready for PCR amplification. The PCR reaction mix was set up with a 20 µl final volume and contained the following components for each sample:
Final Component Volume Concentration
REDExtract-N-Amp PCR Reaction Mix (2x) 10 µl 1x
SPR2-Geno-WT/KO-Forward Primer; Pr. 1 (10 mM) 0.8 µl 400 nM
SPR2-Geno-WT-Reverse Primer; Pr. 2 (10 mM) 0.8 µl 400 nM
SPR2-Geno-KO-Reverse Primer; Pr. 3 (10 mM) 0.8 µl 400 nM
Tissue Extract 4 µl
Sterile Water 3.6 µl
Final Volume 20 µl
WT and KO PCRs were performed in the same tube using the following primers (Figure 13 A, Appendix 9.1.):
Primer Sequence
SPR2-Geno-WT/KO-Forward Primer, Pr. 1 5` - GCTTGACCGGCACCCCGGTGAG - 3`
SPR2-Geno-WT-Reverse Primer, Pr. 2: 5` - GGGCACTGGATCCCTTGGAGC – 3`
SPR2-Geno-KO-Reverse Primer: Pr. 3: 5` - CGGATCTCAAACTCTCCTCC - 3`
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4. Materials and Methods
The REDExtract-N-Amp PCR Reaction Mix already contains buffer, salts, dNTPs, Taq polymerase, JumpStart Taq antibody for hot start, and REDTaq loading dye. WT and KO fragments differing in size were amplified from the isolated DNA using the following cycling program:
Step Temperature Time Number of Cycles
Initial Denaturation 94°C 3 min 1
Denaturation 94°C 1 min
Annealing 59°C 1 min 30
Extension 72°C 1 min
Final Extension 72°C 10 min 1
The resulting 803 bp WT fragment and the 452 bp KO fragment were separated on an agarose gel containing 1% agarose in 1 x TAE buffer (1 x Tris-Acetate-EDTA buffer: 40mM Tris, 20mM acetic acid, 1mM EDTA = ethylenediaminetetraacetic acid) and ethidium bromide at a final concentration of 0.5 µg/ml. PCR products were visualized, identified, and photographed by putting the gel on a UV-transilluminator (Gel Logic 100 Imaging System, Kodak) and by comparing the sizes of the PCR products with a DNA ladder (GeneRuler™ DNA Ladder Mix, Thermo Scientific; Cat. No. SM0331).
4.2. Mouse Experiments
In this study, all mouse experiments were conducted using at least seven SPRED2 KO mice and seven WT littermates as control. The WT and KO mouse groups always contained mice of comparable ages and comparable amounts of female and male littermates. The ages of mice used for the studies ranged between four and 24 months. The exact numbers and average ages of the mice used in the respective experiments are indicated in the “Results” section. All experiments have been approved by the local councils for animal care and were performed in accordance to the European law for animal care and use.
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4. Materials and Methods
4.2.1. Examination of Water Uptake
Mice were kept in separate cages under standard conditions and were habituated to the new environment for 48 h. Water bottles were weighed every day for the period of one month and the volumes of consumed water were noted. The sum of the daily water volumes divided by the number of days revealed the average daily water uptake. Due to the hypochondroplasia-like dwarfism and the thereof dependent significantly reduced body weight in the SPRED2 KOs, the daily water consumption was further calculated in relation to body weight. Therefore, average daily water uptakes were divided by the respective body weight.
4.2.2. Experimental Water Deprivation
Mice were kept in standard cages individually or in groups according to their habitual environment. Water deprivation was carried out for 48 h, while mice had free access to mouse chow. A control group of animals was kept having free access to mouse chow and water. After 48 h both water-starved and control mice were weighed, trunk blood was taken, and serum was prepared as described below (4.2.3). Serum osmolalities were determined via freezing-point depression (Osmomat 030, Gonotec). For calculation of organ/body weight ratios of brain, adrenal gland, and kidney, total weights of tissues were measured and divided by the corresponding body weights.
4.2.3. Blood Collection and Serum Preparation
For routine laboratory measurements and most hormone determinations, blood was collected from the retro-orbital plexus. Therefore, mice were anesthetized by intraperitoneal injection of avertin (250 mg/kg body weight). Capillaries were generated by cutting the tips of Pasteur pipettes into pieces of 1 cm length with a glass knife. They were stored in PlusOne Repel-Silane ES (GE Healthcare; Cat. No. 17-1332-01), which prevents blood clotting within the capillary. For determination of serum AVP, trunk blood was collected in serum tubes after slight anesthetization with isoflurane (Isofluran CP, CP Pharma, Cat. No. 798-932). Blood was clotted in the serum tubes for 1 h. Coagulated blood was centrifuged at 3.000 x g for 15 min. The serum in the supernatant was stabilized by protease inhibitors (Complete EDTA-free Protease Inhibitor Cocktail Tablets, Roche, Cat. No. 04693132001), aliquoted, and stored at -80°C. Serum samples were used for determination of all in “Table 1” indicated routine serum parameters and for all hormone determinations except of CRH.
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4. Materials and Methods
Preparation of Avertin
Stock Solution (1g/ml) 1.25 g 2,2,2 tribromethanol (Cat. No. T48402) 1.25 ml tert-amyl alcohol (2-methyl-2-butanol, Cat. No. 152463)
Working Solution (25 mg/ml) 0.25 ml Stock Solution 9.75 ml 0.9% saline
4.2.4. Determination of Routine Laboratory Serum Parameters
Serum analysis of routine laboratory parameters was conducted in the Central Core Laboratory of the University Hospital Wuerzburg by standard automated laboratory analyzers using well- established and quality-controlled methods.
4.2.5. Determination of Hormone Levels
For detection of serum ACTH, aldosterone, corticosterone Ang II, GH, and IGF-1 blood was collected from the retro-orbital plexus between 9 and 12 a.m. For determination of serum AVP trunk blood was collected from water-starved and non-starved mice between 1 and 4 p.m. Serum was prepared as described above (4.2.3) and hormonal measurements were performed by EIA (enzyme immunoassay) or ELISA (enzyme-linked immunosorbent assay) using commercially available assays according to the manufacturer’s instructions. CRH was measured in brain lysates and in cell culture supernatants mHypoE-44 cells. For tissue lysates, mouse brains were prepared, the hypothalamic region containing the PVN was punched with a mold, and tissue was homogenized according to the manufacturer’s instructions of the CRH ELISA kit. Hypothalamic lysates were stabilized with protease inhibitors (Complete EDTA-free Protease Inhibitor Cocktail Tablets, Roche, Cat. No. 04693132001), aliquoted, and stored at -80°C. For cell lysates, mHypoE-44 cells were seeded on 96-well plates and transfected with either SPRED1 or SPRED2 expression plasmids or empty vector (4.4.3). After starvation in low glucose DMEM without FBS for 48 h (4.4.5.), cell culture supernatants of mHypoE-44 cells were pipetted off and 50 µl of supernatants was used for CRH determination. In each assay, determination of hormone levels in both provided standard reagents and mouse samples were performed in duplicate. The optical absorbance, which reflects the amount of hormone contained in the sample, was measured on a microplate reader (Wallac Victor2 1420 Multilabel Counter, Perkin Elmer) at a wavelength of 450 nm. The mean values of duplicate readings were calculated for each standard and sample. A standard curve was generated by plotting the standard concentrations on the x-axis and the corresponding mean absorbance at
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4. Materials and Methods
450 nm on the y-axis. For generation of the standard curve and for calculation of the unknown concentrations from the standard curve based on the measured absorptions, a four-parameter curve fitting software was used (Prism 4, GraphPad Software).
Hormone Assay Company
Aldosterone ELISA ALPCO Diagnostics, Cat. No. 11-ALDHU-E01
Corticosterone EIA Diagnostic Systems Laboratories, Inc., Cat. No. DSL-10-81100
ACTH EIA Peninsula Laboratories, LLC, Cat. No. S-1130
Ang II EIA Peninsula Laboratories, LLC, Cat. No. S-1133
GH ELISA Millipore, Cat. No. EZRMGH-45K
IGF-1 ELISA Assay Pro, Cat. No. EMI1001-1
AVP EIA Assay Designs, Cat. No. 900-017
CRH ELISA Yanaihara Institute Inc., Cat. No. YK131
4.2.6. Wound Control and Monitoring
Mice that developed bloody and purulent wounds in facial and neck areas due to the excessive grooming behavior were controlled twice a week. Mice showing severe wounds, which extended the size of 1 cm2 and did not improve within two weeks, were euthanatized or immediately used for experiments. In order to be able to monitor wound development, lesions were photographed by a digital camera (Coolpix 995, Nikon) once in a week.
4.3. Cloning of cDNAs and Expression Plasmids
4.3.1. pcDNA3.1‐EVH1‐β‐geo and pcDNA3.1‐β‐geo
EVH1-β-geo, a fusion protein comprising a truncated form of SPRED2 and the gene trap vector- included reporter gene β-geo (fusion gene composed of β-galactosidase gene (lacZ) and neomycin resistance gene (neoR)), is still expressed in our SPRED2 KO mice. EVH1-β-geo contains a functional EVH1 domain but is without any attributed function yet. Both the EVH1-β- geo fusion protein and the β-geo reporter, the latter acting as a negative control for further 52
4. Materials and Methods experiments, were cloned directly from RNA of SPRED2 KO mice (Appendix 9.2.). Because SPRED2 is highly expressed in brain and neural tissues, we used whole brain for RNA extraction. 1 ml of Trizol (Invitrogen, Cat. No. 10296-010) was added to 100 mg of brain tissue, and RNA was extracted from brain according to the manufacturer’s instructions. For RT-PCR, 2 µg of total RNA and the following primers, which included the required restriction sites for cloning into pcDNA3.1/myc-His(-) B (NotI/HindIII), were used. Reverse transcription and amplification were performed using the Verso 1-Step RT-PCR Kit (Thermo Scientific, Cat. No. AB-1454/A) and a standard PCR protocol suggested by the manufacturer.
Primer Sequence
NotI-S2/EVH1-β-geo -Forward, Pr. 4 5´-TAGCGGCCGCATGACCGAAGAAACACACCCGG-3´
NotI-β-geo-Forward, Pr. 5: 5´-TAGCGGCCGCGTCCCAGGTCCCGAAAACCAAA-3´
HindIII-Reverse (for both), Pr. 6: 5´-ATAAGCTTTCAGAAGAACTCGTCAAGAAGG-3´
After separation of PCR products by agarose gel electrophoresis (4.1.3.), DNA fragments of the predicted sizes, i.e. 4,422 bp for EVH1-β-geo and 3,984 bp for β-geo, were identified on a UV- transilluminator, cut out of the gel, and eluted using the NucleoSpin® Gel and PCR Clean-up Kit (Macherey-Nagel, Cat No. 740609.50). The purified cDNAs were cloned into the pCR-XL- TOPO® cloning vector via T/A cloning, which was enabled by their A-overhangs on 3´DNA ends produced by Taq polymerase and via the T-overhangs provided on 3´DNA ends of the pCR-XL- TOPO® cloning vector (TOPO® XL PCR Cloning Kit, Invitrogen, Cat No. K4700-10). After transformation of pCR-XL-TOPO®-EVH1-β-geo and pCR-XL-TOPO®-β-geo plasmids into One Shot® TOP10 Electrocomp™ E. coli (Invitrogen, included in TOPO® XL PCR Cloning Kit, Invitrogen, Cat No. K4700-10) and low scale plasmid preparation from bacteria cultures supplemented with suitable antibiotics (NucleoSpin® Plasmid Kit, Macherey-Nagel, Cat. No. 740588.250), the integrity and sequence of the EVH1-β-geo and β-geo inserts were checked by appropriate analytical restriction digests and custom DNA sequencing (Eurofins MWG; Ebersberg). Consecutively, pcDNA3.1/myc-His(-) B vector (Invitrogen, Cat. No. V855-20), a plasmid designed for overproduction of proteins in mammalian cell lines, pCR-XL-TOPO®- EVH1-β-geo and pCR-XL-TOPO®-β-geo plasmids were cut with NotI and HindIII in a preparative restriction digest (Fermentas). The desired DNA fragments, i.e. the digested backbone of pcDNA3.1/myc-His(-) B cloning vector and the digested inserts EVH1-β-geo and β- geo, were again eluted from an agarose gel after gel electrophoresis. Via ligation of sticky ends (Quick Ligation™ Kit, New England Biolabs, Cat. No. M2200S), EVH1-β-geo and β-geo were cloned into the NotI/HindIII sites of pcDNA3.1/myc-His(-) B. Both pcDNA3.1-EVH1-β-geo and 53
4. Materials and Methods pcDNA3.1-β-geo expression plasmids were amplified in and re-isolated from bacterial cultures supplemented with appropriate antibiotics. Repeated analytical restriction digests and custom DNA sequencing verified the integrity of pcDNA3.1-EVH1-β-geo and pcDNA3.1-β-geo plasmids so that they were produced in large scale (NucleoBond® PC 500 Kit, Macherey-Nagel, Cat. No. 740574) for the transfection and expression in eukaryotic cells.
4.3.2. pGL3‐CRHProm‐RE and pGL3‐CRHProm
In order to perform in vitro assays to monitor the activity of the CRH promoter, we cloned a 5.7 kb region of the CRH promoter, which contained exon 1, critical regulatory elements, and transcription factor binding sites (Appendix 9.3.). In particular, the CRH gene contains a repressor element-1/neuron-restrictive silencing element (RE-1/NRSE) in the intron between CRH exons 1 and 2, a critical regulatory element, which is able to silence CRH transcription (Seth and Majzoub, 2001). In order to exclude possible suppressive effects of RE-1/NRSE on CRH transcription in general, we cloned both the CRH promoter region with and without the RE- 1/NRSE repressor element (Figure 30 A and B). We amplified the 5.7 kb CRH promoter sequence including the RE-1/NRSE repressor (Figure 30 A) by PCR from genomic mouse DNA. DNA was isolated from thymus of a WT mouse using the DNeasy Blood & Tissue Kit (Qiagen, Cat. No. 69506). For PCR, 150 ng of isolated genomic DNA and the following primers including required restriction sites for cloning into pGL3-Basic vector (XhoI) were used. Amplification was conducted via Phusion High-Fidelity DNA Polymerase (Thermo Scientific, Cat. No. F-530S) according to a standard PCR protocol suggested by the manufacturer.
Primer Sequence
XhoI-CRH-RE-1/NRSE-Forward, Pr. 7 5´-CCGCTCGAGATGGCCTTTCCAAGGGTAATTC-3´,
XhoI-CRH-RE-1/NRSE-Reverse, Pr. 8: 5´-CCGCTCGAGAGAAGGTGGGGGAGAAAGGTAA-3´
The DNA fragment representing the CRH promoter sequence including the RE-1/NRSE element with the predicted size of 5,834 bp was first cloned into the pCR-XL-TOPO® cloning vector in analogy to the cloning procedure indicated above in 4.3.1. Because the Phusion High- Fidelity DNA Polymerase does not produce A-overhangs on 3´DNA ends, A-tailing was performed using the PCR product purified from the agarose gel. After incubating with DreamTaq DNA Polymerase (Thermo Scientific, Cat. No. EP0703) for 20 min at 72°C, the A-tailed cDNA ® was again purified from an agarose gel. From pCR-XL-TOPO -CRHProm-RE, the cloned CRH promoter region was transferred into the pGL3-Basic vector, which contains a firefly luciferase
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4. Materials and Methods
® reporter gene (Promega, Cat. No. E1751). Both pCR-XL-TOPO -CRHProm-RE and pGL3-Basic were cut with XhoI (Fermentas). pGL3-Basic was further dephosphorylated on 5`ends using Antarctic Phosphatase (New England Biolabs, Cat.No. M0289S) in order to avoid vector re- ligation. Via ligation of sticky ends, CRHProm-RE was cloned in front of the luciferase reporter gene in the pGL3-Basic vector, which allows the monitoring of reporter gene expression under control of the CRH promoter after transfection into eukaryotic cell lines. To be able to study CRH promoter activity also in the absence of the RE-1/NRSE repressor, we generated a second CRH promoter reporter without the RE-1/NRSE element (Figure 30 B). Because we were not able to amplify and to clone the shortened CRH promoter region directly from genomic mouse DNA, the RE-1/NRSE silencer was removed retroactively, resulting in a CRH promoter sequence with a predicted size of 5,669 bp. The only endonuclease with the property to cut once between exon 1 and RE-1/NRSE in the CRH promoter and to not cut in the pGL3-Basic vector was the restriction endonuclease Eco91I (Fermentas). Eco91I further fitted in a double digest with BglII or HindIII, the only usable restriction sites in the pGL3-Basic vector for our purpose. Because the double digest of pGL3-CRHProm-RE with Eco91I and BglII produced sticky ends that did not fit to each other, T4 DNA Polymerase (Thermo Scientific, Cat. No. EP0061) was used to blunt DNA ends due to its ability to fill-in 5' DNA-overhangs and to remove 3' DNA-overhangs. After incubation with T4 DNA Polymerase according to the manufacturer’s instructions, quick ligation of blunt ends was performed, revealing at least a pGL3-Basic-CRH promoter reporter vector lacking the RE-1/NRSE repressor (pGL3-CRHProm).
4.4. Cell Culture, Transfection, and in vitro Assays
4.4.1. Cultivation of HEK 293 T cells
Cells of the adherent HEK 293 T cell line (Human Embryonic Kidney Cells) were grown in high glucose Dulbeccos modified Eagle´s medium (DMEM, Invitrogen, Cat. No. 31966047), which already contained 25 mM glucose, 5.67 mM L-glutamine, and 1 mM sodium pyruvate. Culture medium was further supplemented with 10% fetal bovine serum (FBS, Invitrogen, Cat. No.
10500-064). To maintain the cell line, cells were kept on 10 cm plates at 37°C and 5% CO2 and grown in a monolayer to a confluency of 80-90%. Every 3 to 4 days, cells were rinsed in sterile phosphate buffered saline (1xPBS: 137 mM NaCl, 2,7 mM KCl, 10 mM Na2HPO4, 2mM
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4. Materials and Methods
KH2PO4, pH 7.4), detached with 0.05% trypsin-EDTA (Invitrogen, Cat. No. 15400054), and replated at a ratio of 1:10 or 1:20 after resuspension in fresh culture medium.
4.4.2. Cultivation of mHypoE‐44 cells
Cells of the embryonic mouse hypothalamic cell line mHypoE-44 also grew in monolayer cultures attached to the tissue culture plate. They were maintained in high glucose DMEM (Invitrogen, Cat. No. 61965059), which already included 25 mM glucose and 3.97 mM L- glutamine and which was supplemented with 10% FBS. For cultivation, mHypoE-44 cells were kept on 10 cm plates at 37°C and 5% CO2 until the adherent monolayer reached a confluency of 70-90%. Every 2 to 4 days, cells were rinsed with 1xPBS, detached with 0.05% trypsin-EDTA, and splitted with a plate ratio of 1:4 to 1:10 in fresh culture medium.
4.4.3. Transfection of HEK 293 T and mHypoE‐44 cells
24 h before transfection, HEK 293 T and mHypoE-44 cells, respectively, were splitted in an appropriate ratio and seeded in the appropriate culture medium to reach 90% confluency overnight. Cells were transiently transfected with Lipofectamine® 2000 Reagent (Invitrogen, Cat. No. 11668-027). The used amount of DNA, culture medium, and transfection reagent depended on the size of culture plates, as also indicated in the manufacturer’s instructions. Transfection of HEK 293 T and mHypoE-44 cells was performed with the appropriate culture media without FBS. Dependent on the experimental design, cells were single or cotransfected with the expression plasmids listed below (Appendix 9.4., Vector Cards):
Name of Plasmid Vector Backbone cDNA
Mock pcDNA3.1/myc-His(-) A -
Mock pcDNA3.1/myc-His(-) B -
Mock pGL3-Basic -
pcDNA3.1-SPRED1 pcDNA3.1/myc-His(-) A Spred1
pcDNA3.1-SPRED2 pcDNA3.1/myc-His(-) A Spred2
pcDNA3.1-EVH1-β-geo pcDNA3.1/myc-His(-) B EVH1-β-geo
pcDNA3.1-β-geo pcDNA3.1/myc-His(-) B β-geo
pRL-TK pTK Renilla Luciferase
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4. Materials and Methods
pGL3-CRHProm-RE pGL3-Basic CRHProm-RE
pGL3-CRHProm pGL3-Basic CRHProm
pGL2-4xEts1 pGL2-Basic HSV-TK-4xEts1
6 h after transfection, serum-free medium was replaced by normal culture medium containing FBS. 18 - 48 h after transfection, cells were prepared for Western Blot, ERK activity assay, luciferase assay, ELISA, or X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) staining.
4.4.4. In vitro ERK Activity Assay
24 h before transfection, HEK 293 T cells were seeded on 6-well plates coated with 0.1% gelatin in PBS. Cells were transiently transfected with pcDNA3.1 expression plasmids for SPRED1, EVH1-β-geo, β-geo, or empty vector as described above (4.4.3). After transfection, serum-free medium was not replaced as usual but cells were serum-starved for 36 h in order to downregulate GF-dependent pathways. In order to re-activate especially the Ras/ERK/MAPK cascade in the starved cells, serum-free medium was removed and transfected HEK 293 T cells were stimulated for 3 min with 10 ng/ml EGF (Cell Signaling, Cat. No. 8916). EGF was dissolved in Krebs-Ringer-HEPES buffer (122.5 mM NaCl, 5.4 mM KCl, 0.8 mM MgCl2, 1.2 mM
CaCl2, 1.0 mM NaH2PO4, 10 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 6 mM Glucose), which was pre-warmed to 37°C. After EGF-dependent stimulation of ERK phosphorylation and activity, cells were homogenized for Western blot analyses.
4.4.5. In vitro CRH Promoter Reporter Assay
24 h before transfection, mHypoE-44 cells were seeded on 96-well plates. Cells were either transfected (4.4.3) with the CRH promoter luciferase reporter vector containing the RE-1/NRSE repressor (pGL3-CRHProm-RE) or lacking it (pGL3-CRHProm). Additionally, cells were cotransfected with the Renilla luciferase control reporter vector (pRL-TK, Promega, Cat. No. E2241). CRH promoter activity was either monitored in the presence of cotransfected SPRED1 and SPRED2, respectively, and in each case both in the presence or absence of EVH1-β-geo. In order to stimulate CRH promoter activity, which is normally induced in response to stress, mHypoE-44 cells were starved for glucose and FBS after transfection. Thus, the serum-free but high glucose DMEM was replaced by low glucose DMEM without FBS (5.56 mM glucose, 3.97 mM L-glutamine, Invitrogen, Cat. No. 21885108) for 24 - 30 h after transfection. Firefly and 57
4. Materials and Methods
Renilla luciferase activity were determined using the Dual-Glo System (Promega, Cat No. E2920) and detecting the luminescence by a microplate reader (Wallac Victor2 1420 Multilabel Counter, Perkin Elmer). For each sample, firefly luciferase signals were normalized to Renilla luciferase activity. Because each of the various combinations of plasmid transfections were performed at least twelve times, mean values were calculated from the firefly/Renilla luciferase signal ratios for each experimental approach. The mean value of samples only transfected with CRH promoter luciferase reporter and Renilla luciferase control vector was set to 1 and mean values of all other cotransfected samples were expressed as x-fold of 1.
4.4.6. In vitro Ets Transcription Factor Reporter Assay
According to the CRH promoter reporter assay, an Ets transcription factor reporter assay was performed. Instead of the CRH promoter luciferase reporter (pGL3-CRHProm), a luciferase reporter vector containing 4 Ets1 transcription factor binding sites (pGL2-4xEts1) was used (Appendix 9.5., 4.4.3., (Hoffmeyer et al., 1998)). mHypoE-44 cells were prepared for transfection and transfected as described above (4.4.3., 4.4.5.) with the exception that cells were only starved for FBS but not for glucose 24 - 30 h after transfection. Luciferase activity determination and data analyses were also performed in accordance to the CRH promoter reporter assay (4.4.5.).
4.5. Analysis and Quantification of Protein and mRNA Expression
4.5.1. Western Blot
For analysis of protein expression in cell lysates by Western Blot, cells were seeded on 6-well plates 24 h prior to transfection and transfected with the desired plasmids as described in 4.4.3. 18 - 48 h past transfection or after termination of ERK activity assay, cells were scratched from culture wells and lysed in 2% SDS (sodium dodecyl sulphate) in PBS. Lysis buffer was supplemented with Complete EDTA-free Protease Inhibitor Cocktail Tablets and PhosSTOP Phosphatase Inhibitor Cocktail Tablets (Roche, Cat. No. 04906837001) to prevent degradation and de-phosphorylation of proteins, in our case especially the de-phosporylation of ERK. For
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4. Materials and Methods analysis of mouse tissue lysates, 50 mg of the desired tissue was homogenized with a homogenizer (Polytron PT 3100) in 1 ml of 2% SDS/PBS sample buffer containing protease and phosphatase inhibitors as described above. If cell or tissue samples appeared very viscous due to DNA released from the nucleus, lysates were sonified for 10 s at 50% duty cycle and 50% power (Sonopuls HD 2070, Bandelin). Protein concentrations were estimated via UV- spectroscopy by inserting the measured absorptions into the following formula: protein concentration (mg/ml) = 1.55 x (A280-A320) - 0.76 x (A260-A320). Protein samples that were not immediately used for Western blot were stored at -80°C. Before loading on 6 - 12% polyacrylamide gels, probes were supplemented with Laemmli buffer. Protein samples (cell lysates: 5 - 20 µg, mouse tissue lysates: 30 - 50 µg of total protein) and a marker containing proteins with defined sizes (PageRuler Prestained Protein Ladder, Fermentas, Cat. No. 26616) were separated by SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) under reducing conditions according to their size. The separated proteins were electrotransferred from the gel to nitrocellulose membranes (Protran BA38, Whatman, Cat. No. 10401396) using semi-dry blotters (Fastblot B44, Biometra, 015-200). Protein transfer and loading amount were verified by Ponceau S staining of the nitrocellulose membranes.
Solution/Buffer Contents
® Resolving Gel (10 ml), 5.3 - 3.3 ml H2O, 2 - 4 ml 30% acrylamide mix (Rotiphoprese Gel 30, depending on percentage Roth, Cat. No. 3029.1), 2.5 ml 1.5 M Tris (pH 8.8), 100 µl 10% SDS, (6 - 12%): 100µl 10% ammonium persulfate (APS), 8 - 4 µl tetramethylethylendiamin (TEMED)
Stacking Gel (10 ml, 5%) 6.8 ml H2O, 1.7 ml 30% acrylamide mix, 1.25 ml 1.0 M Tris (pH 6.8), 100 µl 10% SDS, 100µl 10% APS, 10 µl TEMED)
5 x Laemmli Buffer (250 mM Tris pH 6.8, 10% SDS, 40% glycerol, 0.05% bromphenolblue, 5% β-mercaptoethanol)
1 x SDS-PAGE Running Buffer 25 mM Tris base, 192 mM glycine, 0.1% SDS
1 x Western Blot Transfer Buffer 25 mM Tris base, 192 mM glycine, 0.1% SDS, 20% methanol
Ponceau S Staining Solution 2% Ponceau S, 30% trichloracetic acid, 30% sulfosalicylic acid
Wash Solution 0.05% Tween 20 in PBS
Blocking/Antibody 4% non-fat dry milk powder in 0.05% PBS-Tween 20 Incubation Solution
To reduce unspecific antibody binding, membranes were blocked with 4% milk in 0.05% PBS- Tween for at least 1 h. Membranes were incubated with primary antibodies diluted in blocking 59
4. Materials and Methods solution at 4°C overnight and washed 4 times for 5 min with 0.05% PBS-Tween. Incubation with the secondary antibodies was performed at room temperature for 1 - 2 h before blots were washed again. For signal detection, the ECL (Enhanced Chemoluminescence) Plus Western Blotting Substrate Kit was used according to the manufacturer’s instructions (Pierce, Cat. No. PI80196).
Antibody Dilution Company/Reference
SPRED2 1:500 (Bundschu, 2005; Bundschu et al., 2005)
β-galactosidase (β-gal) 1:5,000 Abcam, Cat. No. ab616 p44/42 MAPK (ERK) 1:2,000 Cell Signaling, Cat. No. 9102
Phospho-p44/42 MAPK (P-ERK) 1:1,000 Cell Signaling, Cat. No. 9101
Peroxidase-AffiniPure Goat Anti-Rabbit 1:10,000 Jackson ImmunoResearch, Cat. No. 111-035-144) IgG (Immunoglobulin G)
4.5.2. Northern Blot
Northern blot analysis was performed in order to examine the levels of CRH mRNA in SPRED2 KO mice and WT controls. Mice were decapitated, brains were removed, and the hypothalamic PVN region was prepared by punching with a mold. RNA was isolated using Trizol (Invitrogen, Cat. No. 10296-010) and re-suspended in RNase-free DEPC water. 20 μg of each total RNA sample were supplemented with RNA loading dye (Thermo Scientific, Cat. No. R0641) and size-separated in a formaldehyde-agarose gel. The separated RNA was transferred onto a nylon membrane (Hybond™-N+, GE Healthcare, Cat. No. 45-000-932) by a capillary blot using 20 x SSC and was fixed to the membrane by UV crosslinking for 3 min at 312 nm. To generate a CRH-specific probe, a 357 bp cDNA sequence of CRH located in exon 2 was amplified by RT- PCR (Appendix 9.7., Verso 1-Step RT-PCR Kit, Thermo Scientific, Cat. No. AB-1454/A). Therefore, RNA isolated from a WT mouse brain, the following primers, and a standard RT-PCR protocol suggested by the manufacturer were used.
Primer Sequence
CRH-Ex2-Forward, Pr. 9 5´-AGCCCTTGAATTTCTTGCAG-3´
CRH-Ex2-Reverse, Pr. 10: 5´-GGTGGAAGGTGAGATCCAGA-3´
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4. Materials and Methods
The probe was labeled radioactively with 32P using the Prime-It II Random Primer Labeling Kit (Stratagene, Cat. No. 300385) according to the manufacturer’s instructions. In order to block unspecific binding of nucleic acids to the membrane, the membrane was pre-hybridized in hybridisation buffer for 1 h at 42°C. After purification of the radioactively labeled probe using a Sephadex column (PD MiniTrap G-10, GE Healtcare), the probe was added to the hybridisation solution and incubated on the membrane for 24 h at 42°C. The membrane was washed twice with low stringency wash buffer at 42°C first and then twice with high stringency wash buffer at 55°C. Radiographic signals resulting from the amount of radioactive 32P bound to the Northern blot membrane and representing the expression level of CRH mRNA were detected by a Phosphoimager (BAS-1500, FujiFilm) and quantified as described below (4.5.3.).
Solution/Buffer Contents
DEPC-H2O 0.1% diethylpyrocarbonate (DEPC) in H2O, stirred for at least 2 h at 37°C, then autoclaved to inactivate DEPC
Running Buffer (1 x MOPS) 20 mM MOPS (3-(N-morpholino)propanesulfonic acid), 5 mM sodium acetate, 1 mM EDTA
RNA Gel 1% RNA agarose in 1 x MOPS
20 x SSC (Saline-Sodium Citrate) 3 M NaCl, 0.3 M trisodium citrate
100 x Denhard´s Solution 2% bovine serum albumin (BSA), 2% Ficoll, 2% polyvinylpyrolidone
Hybridisation Buffer 50% formamide, 25% 20 x SSC, 5% 100 x Denhard´s Solution, 5% 10% SDS, 500 µg salmon sperm DNA (Applichem, Cat. No. A2160)
Low Stringency Wash Buffer 2 x SSC, 0.1% SDS
High Stringency Wash Buffer 0.2 x SSC, 0.1% SDS
4.5.3. Quantification of ERK Phosphorylation and of CRH mRNA Levels in Brain
For quantification of ERK activity, Western blots demonstrating the levels of ERK and P-ERK in WT and SPRED2 KO mice were scanned and converted to TIFF files. Band intensities were quantified using ImageJ software (National Institutes of Health) and expressed as arbitrary densitometric units. For every mouse sample, the ratio of P-ERK to total ERK was calculated. Mean value of all WT samples was set to 1 and mean value of KO samples was expressed as x-fold of WT.
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4. Materials and Methods
For quantification of CRH mRNA levels, band intensities were also quantified using ImageJ. For every mouse sample, the ratio of CRH mRNA band intensities to total RNA was calculated, the mean ratio of WT was normalized to 1, and mean value of KO samples was expressed as x-fold of WT.
4.6. Histology and Immunohistochemistry
4.6.1. Hematoxylin and Eosin Staining (H&E Staining)
To investigate morphology, kidneys were dissected from sacrificed mice, fixed in 4% paraformaldehyde (PFA)/PBS for 24 h, and embedded in paraffin. 7 µm sections were collected on SuperFrost Plus microscope slides (Thermo Scientific, Cat No. 10149870). According to a standard protocol (http://web.uni-frankfurt.de/fb15/nrp/br/script/node18.html), sections were deparaffinized, rehydrated, stained with haematoxylin and eosin, dehydrated, and mounted with Entellan (Merck Millipore, Cat. No. 107960). Overview photos were taken by an Axiophot 2 microscope equipped with a 2.5-fold plan objective and an AxioCam MRc Rev.3 FireWire camera (Zeiss). Detailed photos were taken by an Eclipse E600 microscope (Nikon) equipped with 10-, 60-, and 100-fold objectives and a DXM1200F camera (Nikon).
4.6.2. TUNEL Assay
Apoptotic processes in kidneys were investigated by a TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) assay, which was based on the breakdown of DNA especially in apoptotic cells. Dissected kidneys were embedded in Tissue-Tek OCT-compound (Sakura, Cat. No. 4583) in compatible plastic tubes and snap-frozen in liquid nitrogen. 5 µm cryosections were cut using a CM1950 cryostat (Leica) equipped with C35 carbon steel microtome blades (Feather/PFM medical, Cat. No. 207500003) and collected on SuperFrost Plus microscope slides. Slices were fixed, permeabilized, and fragmented DNA of apoptotic cells was labeled with flourescein-12-dUTP at 3´-OH DNA ends using the enzyme terminal deoxynucleotidyl transferase according to the manufacturer’s instructions (DeadEndTM Flourometric TUNEL System, Promega, Cat. No. TB235). Thus, apoptotic cells in the kidney could be identified by green fluorescence. Nuclei of all renal cells were counterstained with 0.1µg/ml DAPI (4',6-diamidino-2-phenylindole) in PBS. All stained sections were mounted with
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4. Materials and Methods
Mowiol 4-88 (Roth, Cat. No. 9002-89-5) and investigated by a BX41 microscope (Olympus) equipped with 20- and 60-fold objectives and a CCD-FV2T camera (Olympus). Photos were pseudo-colored using the computer-assisted image analysis system Cell-D (Olympus).
4.6.3. Immunohistochemistry
Dissected brains, pituitary glands, adrenal glands, and kidneys were embedded in Tissue-Tek, and 10 µm cryosections were prepared as described above (4.6.2.). Tissue slices were fixed in 4% PFA/PBS for 10 min, permeabilized with 0.2% Triton X-100 in PBS for 20 min, and blocked in 10% goat serum/PBS for at least 1 h. Primary antibodies were incubated overnight, secondary antibodies for 2 h, both in 10% goat serum/PBS and in a humidified chamber in the dark. After incubation with antibodies, slides were washed 4 times with PBS. Stained sections were investigated and photographed using an Eclipse E600 microscope equipped with a C1 confocal scanning head and 10-, 60-, and 100-fold objectives (Nikon).
Antibody Dilution Company/Reference
SPRED2 1:50 (Bundschu, 2005; Bundschu et al., 2005)
Aldosterone Synthase 1:50 (Wotus et al., 1998)
Alexa Fluor® 488 Goat Anti-Rabbit IgG 1:1,000 Invitrogen, Cat. No. A11008
Alexa Fluor® 594 Goat Anti-Rabbit IgG 1:1,000 Invitrogen, Cat. No. A11012
4.6.4. X‐Gal Staining
X-Gal stainings were performed as previously described (Ullrich and Schuh, 2009). In brief, HEK 293 T cells were stained upon removal of culture medium and washing with PBS. For staining of tissue sections, 5 µm cryosections were prepared as described above (4.6.2) and slices were mounted with glycerol after staining. Whole organs were washed with PBS and stained directly after dissection. All specimens were fixed in X-Gal Fixation Buffer. Cells and tissue sections were fixed for 15 min, organs for 15 min up to 1 h depending on size. After washing of cells and slices with X-Gal Wash Buffer 2 times for 5 min, and of whole organs 3 times for 15 - 30 min, specimens were stained overnight with X-Gal Staining Buffer in a humidified chamber at 37°C. For better permeabilization of organs, 0.01% deoxycholate and 0.02% Nonidet P-40 were included in both wash and staining buffers. Whole organs were photographed by a digital camera (Coolpix 995, Nikon), cells and tissue sections by an Eclipse
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4. Materials and Methods
E600 microscope equipped with 10-, 60-, and 100-fold objectives and a DXM1200F camera (Nikon).
Solution/Buffer Contents
0.1 M Phosphate Buffer 27 mM NaH2PO4, 73 mM Na2HPO4
X-Gal Fixation Buffer 0.1 M Phosphate Buffer supplemented with 5 mM EGTA (ethylene
glycol tetraacetic acid, pH 8), 2 mM MgCl2, and 0.2% glutaraldehyde
X-Gal Wash Buffer 0.1 M Phosphate Buffer supplemented with 2 mM MgCl2
X-Gal Stock Solution (50 mg/ml) 500 mg X-Gal in 10 ml dimethylformamide
X-Gal Staining Buffer 0.1 M Phosphate Buffer supplemented with 2 mM MgCl2, 5 mM
K4Fe(CN)6, 5 mM K3Fe(CN)6, and 1mg/ml X-Gal
4.7. Statistics
All results included in this study are expressed as mean standard deviation. Values of p<0.05 were considered as statistically significant and degrees of statistical significance were defined as *p<0.05, **p<0.01, and ***p<0.001.
4.7.1. Basic Statistics
All data sets were analyzed by comparing the different sample groups in each experiment using unpaired two-sample t-tests or Welch´s tests depending on their homogeneity of variances. Normal distribution of data was ensured using graphical analysis tools provided by Statistica (StatSoft), such as box plot and quantile-quantile plot.
4.7.2. Kaplan‐Meier Survival Analysis
To determine the survival probability of SPRED2 KO mice in comparison to WT littermates, we observed 20 SPRED2 KO mice and 20 WT littermate controls starting from their date of birth until the age of 18 months and documented, if applicable, the age of death. Kaplan-Meier
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4. Materials and Methods survival curves were generated using WinSTAT (Statistics Software Add-In for Microsoft Excel). This software included the generation of survival curves and life tables of WT and KO mice, and the calculation of statistical differences between the survival rates of WT and KO groups by computation of logrank test, chi-square, and p values.
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Results 5
5.1. Generation of SPRED2 KO Mice
The family of SPRED proteins was detected by a yeast two hybrid screen and was described by a Japanese group due to their sequence homology to Sprouty proteins (Wakioka et al., 2001). In parallel, our group discovered the SPREDs because of their sequential homology to the EVH1 domain of VASP. To investigate physiological consequences of SPRED deficiency, we started to generate SPRED2 KO mice by a gene trap approach in 2003 (Bundschu, 2005; Bundschu et al., 2005). In brief, the embryonic stem cell clone XB228 originating from 129P2/OlaHsd mice was injected into isolated blastocysts of super-ovulated and fertilized C57BL/6 female mice. The blastocysts were reimplanted into pseudo-pregnant foster mothers that have been mated with vasectomized male mice. Chimeric male offspring were mated to female C57BL/6 WT mice to test for germline transmission and to obtain first SPRED2 HET mice to establish the SPRED2 KO mouse line (Figure 6). The XB228 cell line comprises a disrupted Spred2 gene resulting from the random insertion of the pGT0pfs gene trap vector between exons 4 and 5 of Spred2 (Figure 12 A). This gene trap vector includes a promoterless fusion gene (β-geo). It is composed of a reporter gene (lacZ) allowing profiling of endogenous promoter activity and of an antibiotic resistance gene (neoR) enabling the positive selection of gene trap vector integration. This fusion gene is flanked by an upstream splice acceptor (SA) and a further upstream intronic sequence of the Engrailed2 gene (En2 intron 1). Both the splice branch site included in En2 intron 1 and the SA are required for proper vector integration and splicing. β-geo is flanked downstream by a transcriptional termination sequence (Simian virus 40 polyadenylation site, pA) and by a part of the pUC vector backbone (pUC), which is needed for proper gene trap vector amplification before integration into the mouse genome. Prior to blastocyst injection of the ES cell clone, gene trap vector insertion and expression of the reporter protein β-gal was confirmed by X-Gal staining of ES cells (Figure 12 B). The enzyme β- 67
5. Results gal converts X-Gal to 5,5'-dibromo-4,4'-dichloro-indigo, an intensely blue and insoluble product, which stains cells and tissues and thus monitors Spred2 promoter-driven expression of the reporter instead of Spred2.
Figure 12: Generation of SPRED2 KO Mice - Localization and Verification of Gene Trap Vector Insertion A) The pGT0pfs gene trap vector disrupts the Spred2 gene by random insertion between exons (Ex) 4 and 5. It comprises a part of the Engrailed2 intron 1 structure (En2 intron 1), a splice acceptor site (SA), a promoterless β- galactosidase (lacZ)/neomycin resistance (neoR) fusion gene (β-geo), a Simian virus 40 polyadenylation sequence (pA), and a pUC backbone sequence. A reverse primer located in the gene trap vector (Pr. B) and different forward primers (Prs. A) covering the complete intron 4 of Spred2 were used for definition of the exact gene trap vector insertion point in Spred2 by PCR. For verification of single gene trap vector integration by Southern Blotting, genomic mouse DNA was digested with Bgl II, as indicated cutting only once at the 5´end of the gene trap vector and just upstream of it. Digested DNA samples were labeled using a part of the En2 intron 1 sequence as a probe. Figure based on (Bundschu et al., 2005). B) X-Gal staining of cultured XB228 ES cells demonstrating by blue color the gene trap vector integration in Spred2 and the ability to monitor Spred2 promoter-driven expression of the β-geo reporter gene instead of Spred2. Figure in part reproduced from (Bundschu et al., 2006a) with permission from Elsevier. C) Southern blot with DNA of SPRED2 WT, HET, and KO mice. The upper band, which is present in all samples, corresponds to the endogenous En2 gene. The lower band was only seen in HET and KO mouse DNA and represents the single insertion of the gene trap vector (M: DNA-Marker). Figure in part reprinted from (Bundschu et al., 2005).
The exact gene trap vector insertion point in Spred2 was defined by PCR using a reverse primer located in the gene trap vector and different forward primers covering the complete intron 4 of the Spred2 gene (Prs. A and B, Figure 12 A). Subsequent sequencing of PCR products, which were only obtained with forward primers very near by the insertion point, identified the latter. Single integration was verified by Southern blot with genomic DNA digested with BglII,
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5. Results cutting just upstream of the gene trap vector and only once at its 5´end. A part of the pGT0pfs vector downstream of the BglII cutting site, representing a part of the En2 intron 1 sequence, was used as a radioactive probe (Figure 12 C). The successful disruption of the Spred2 gene in the SPRED2 KO mice was confirmed on RNA level by RT-PCR and Northern blot and on protein levels by Western blot. Hence, we could monitor SPRED2 expression in a great variety of organs isolated from SPRED2 KO mice and WT littermates (Bundschu et al., 2005).
5.2. Breeding and Genotyping of SPRED2 KO Mice
Figure 13: Genotyping of SPRED2 KO Mice A) Genomic exon/intron organization of the murine WT Spred2 gene and of the “trapped” Spred2 gene in SPRED2 KO mice. Offspring was genotyped by PCR using a common forward primer located upstream of the gene trap vector insertion point in intron 4 (SPR2-Geno-WT/KO-Forward Primer; Pr. 1). The WT allele was specifically amplified by combining the forward primer with a reverse primer located downstream of the insertion at the end of intron 4 (SPR2- Geno-WT-Reverse Primer; Pr. 2). For amplification of the KO allele, a reverse primer (SPR2-Geno-KO-Reverse Primer, Pr. 3) located at the beginning of the gene trap vector was used (Ex= Exon, En2 intron 1 = Engrailed2 intron 1 structure, SA = splice acceptor site, β-geo = β-galactosidase (lacZ)/neomycin resistance (neoR) fusion gene, pA = Simian virus 40 polyadenylation sequence, pUC = pUC vector backbone sequence). Figure based on (Bundschu et al., 2005). B) Exemplary picture of a genotyping PCR after agarose gel electrophoresis. According to the primers used for genotyping, WT mice were identified by the amplification of a 803 bp DNA fragment, SPRED2 KOs by the amplification of a 452 bp fragment, and HETs by containing both the WT and KO amplicons (M=Marker).
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5. Results
To maintain the SPRED2 KO mouse line, usually one SPRED2 HET male mouse was mated with two SPRED2 HET female mice. Thus, the offspring contained littermates of all genotypes, i.e. SPRED2 WT, KO, and HET mice with equal ages. Mating of SPRED2 KO mice among each other was also possible, but offspring was obtained in only about 50% of the breeding pairs. To minimize possible inbred effects, mice were kept on a mixed 129P2/OlaHsd C57Bl/6 genetic background. Offspring were genotyped by PCR analysis with a modified and renewed protocol, which allows the amplification of WT and KO alleles together in one reaction and which produces considerably shorter amplicons, thus enabling fast and easy genotyping. For PCR, a common forward primer located upstream of the gene trap vector insertion point in intron 4 (SPR2-Geno- WT/KO-Forward Primer; Pr. 1) was used. In combination with a WT allele-specific reverse primer (SPR2-Geno-WT-Reverse Primer; Pr. 2) located downstream of the insertion at the end of intron 4 and with a KO allele-specific reverse primer (SPR2-Geno-KO-Reverse Primer, Pr. 3) located within the gene trap vector (Figure 13 A, Appendix 9.1.), characteristic WT and KO fragments were amplified. This genotyping PCR results in the generation of an 803 bp WT amplicon and of a 452 bp SPRED2 KO amplicon (Figure 13 B).
5.3. The EVH1‐β‐geo Fusion Protein Expressed in SPRED2 KO Mice May Act Dominant Negative
In our KO mice, the Spred2 gene was disrupted by insertion of a gene trap vector between exons 4 and 5 of Spred2 (Figure 12 and 13). Although the Spred2 gene was interrupted by gene trap vector insertion, the first 4 exons of Spred2 remained intact. Because of the SA and the splice branch site provided by the gene trap vector and due to the SD in upstream exon 4, β-geo is spliced to the end of Spred2 exon 4 after transcription of the trapped Spred2 allele. Upon translation of the “trapped” Spred2 gene, either the sole β-geo reporter is expressed if the internal start codon of β-geo is used or a fusion protein harboring Spred2 exon 1-4 and β-geo if the start codon of Spred2 is used (Figure 14). Thus, a potentially expressed fusion protein would then comprise a complete and functional EVH1 domain encoded by exons 1-3, while KBD and SPR domains, both encoded by exon 6, would be deleted.
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Figure 14: Schematic Illustration of Possible Forms of β-geo Reporter Gene Expression in SPRED2 KO Mice In our SPRED2 KO mice, Spred2 was disrupted by insertion of the gene trap vector pGT0pfs in sense orientation into intron 4 between Spred2 exons 4 and 5. The gene trap vector provides a splice branch site and a SA for the next upstream located exon 4, which itself carries a splice donor site (SD). After transcription, β-geo is spliced to the end of Spred2 exon 4. If protein translation starts at the start codon of Spred2 exon 1 (first bent arrow), a fusion protein of a truncated SPRED2 comprising exons 1-4 and the β-geo reporter is expressed in the SPRED2 KO mice. If protein translation starts at the start codon of the β-geo gene (second bent arrow), only the β-geo reporter is expressed. Independent of translational start, translation stops at the stop codon of β-geo (*). Expression of the fusion protein is inevitably associated with the expression of the complete SPRED2-EVH1 domain in the SPRED2 KOs. For the conduction of in vitro studies, EVH1-β-geo and β-geo, respectively, were subcloned using the indicated primers (Pr. 4, 5, and 6, arrows, Ex = Exon, En2 intron 1 = Engrailed2 intron 1 structure, SA = splice acceptor site, β-geo = β- galactosidase (lacZ)/neomycin resistance (neoR) fusion gene, pA = Simian virus 40 polyadenylation sequence, pUC = pUC vector backbone sequence). Figure modified from (Ullrich et al., 2011).
5.3.1. Deletion of SPRED2 but Expression of an EVH1‐β‐geo Fusion Protein
In order to examine if the EVH1-β-geo fusion protein was present in our SPRED2-deficient mice, we performed Western blot analyzes with brain and kidney lysates, both tissues with confirmed high SPRED2 expression. For protein detection, we used either an anti-SPRED2 antibody or an anti-β-gal antibody. The anti-SPRED2 antibody recognizes an epitope in the coding sequence of exon 4 and hence is able to detect the common SPRED2 isoform but also the EVH1-β-geo fusion protein (Bundschu et al., 2005). The anti-β-gal antibody recognizes the β-geo reporter but also the fusion protein (Figure 15 A). Expression of the common 46 kDa SPRED2 isoform was confirmed by SPRED2-specific antibodies in lysates of WT brain, whereas a prominent 30 kDa band was observed in WT kidney lysates, most likely representing a Spred2 splice variant. Both proteins were absent in the corresponding KO lysates (Figure 15 A, middle panel). Instead, anti-SPRED2 antibodies recognized a 150 kDa protein in the KO lysates. This migrated with the calculated molecular mass of the EHV1-β-geo fusion protein (134 kDa β-geo and 16 kDa N-terminal SPRED2; Figure 71
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15 A, upper panel) and was absent in WT lysates. Mock and Spred2 transfected HEK 293 T cells saved as negative and SPRED2 positive controls, respectively. Anti-β-gal antibodies also detected the 150 kDa band, thereby confirming the expression of the EVH1-β-geo fusion protein in KO mouse organs. In the SPRED2 KO lysates, they additionally recognized a second protein with apparent molecular weight of about 135 kDa, which is consistent with the calculated mass of the β-geo protein and was absent in blots probed with anti-SPRED2 antibodies (Figure 15 A, lower panel). In sum, we could show that translation took place in vivo from both possible start codons, leading to an expression of both the β-geo reporter and the EVH1-β-geo fusion protein in our SPRED2 KO mice.
5.3.2. Functional Expression of Subcloned EVH1‐β‐geo in Eukaryotic Cells
Figure 15: Verification of EVH1-β-geo and β-geo Protein Expression in vivo and in vitro A) Western blot analysis with brain and kidney lysates of SPRED2 KO mice and WT controls. Blots were probed with anti-SPRED2 (upper and middle panel) and with anti-β-gal antibodies (lower panel). In WT tissues, the common 46 kDa isoform of SPRED2 was expressed in brain, while a shortened 30 kDa isoform was expressed in the kidney (middle panel). In KO tissues, both SPRED2 protein isoforms were absent (middle panel); however, the expression of the EVH1-β-geo fusion protein was detected by both anti-SPRED2 and anti-β-gal antibodies (upper and lower panel). Anti-β-gal further detected the expression of the sole β-geo reporter, which was absent in WT tissues and not recognized by anti-SPRED2 antibodies in SPRED2 KO tissues (upper and lower panel). SPRED2 and mock- transfected cells served as positive and negative controls, respectively. B) Western blot analysis with subcloned EVH1-β-geo and β-geo in transfected HEK 293 T cells also demonstrated the expression of both proteins. Anti- SPRED2 antibodies detected specifically the expression of EVH1-β-geo (upper panel), anti-β-gal antibodies both the EVH1-β-geo fusion protein and the β-geo reporter (lower panel). SPRED2 and mock-transfected cells served as positive and negative controls, respectively (middle panel). Figure modified from (Ullrich et al., 2011).
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To get an impression if the expression of the EVH1-β-geo fusion protein or if sole β-geo exerts any effect in vivo and in order to be able to investigate possible effects in vitro, we subcloned both genes from our SPRED2 KO mice (Appendix 9.2.). Therefore, we amplified EVH1-β-geo and β-geo by RT-PCR using RNA isolated from brain of a SPRED2 KO mouse. Forward primers contained the start sequences of Spred2 (Pr.4) and β-geo (Pr.5), respectively, together with an upstream HindIII site for cloning. The common reverse primer (Pr.6) comprised the end of the β-geo sequence with the stop codon and a downstream NotI cloning site (Figure 14). The received cDNAs were subcloned into NotI/HindIII sites of pcDNA3.1/myc-His(-) B vector and transfected in HEK 293 T cells. Expression of both proteins was shown by Western blot using anti-SPRED2 and anti-β-gal antibodies. While anti-SPRED2 incubation revealed only EVH1-β-geo expression (Figure 15 B, upper panel), the anti-β-gal antibody again detected both EVH1-β-geo and β-geo (lower panel). Mock and Spred2 transfected HEK 293 T cells were used as negative and SPRED2 positive controls, respectively (middle and lower panel). To confirm the enzymatic activity of EVH1-β-geo and β-geo (Figure 16 A and B), HEK 293 T cells were transfected with each expression plasmid and stained with X-Gal. In contrast to the mock-transfected control (Figure 16 C), the conversion of X-Gal to its indigo derivative by both proteins was seen by blue staining. Taken together, both the proper expression and the enzymatic functionality of EVH1-β-geo and β-geo proteins could be verified.
Figure 16: Validation of Enzymatic Functionality of Subcloned EVH1-β-geo/β-geo by X-Gal Stainings X-Gal stainings of transfected HEK 293 T cells confirmed the enzymatic activity of A) the EVH1-β-geo fusion protein and B) the β-geo reporter as seen by intense blue staining of cells. C) Mock-transfected cells served as negative control. Scale bars: 100 µm. Figure in part adopted from (Ullrich et al., 2011).
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5.3.3. Putative Dominant Negative Effect of EVH1‐β‐geo on SPRED1 and Ras/ERK/MAPK Signaling in SPRED2 KO Mice
Because SPREDs have been described as MAPK pathway inhibitors, we aimed to examine if EVH1-β-geo or β-geo in our SPRED2 KO mice exerts any effect on function or expression of the remaining SPRED1 and hence on MAPK signaling. Due to the cloning of EVH1-β-geo and β-geo expression constructs, we were able to in vitro reconstitute the in vivo situation of our SPRED2 KO mice, which lack SPRED2 expression but are still capable to express SPRED1. Therefore, we transfected HEK 293 T cells with different expression plasmids in the following manner (Figure 17): either with SPRED1 or with SPRED1 and EVH1-β-geo or with SPRED1 and β-geo. We tested two different ratios of SPRED1 and EVH1-β-geo and β-geo, respectively: EVH1-β-geo/β-geo were either expressed in deficit (3 µg pcDNA3.1-SPRED1 + 3 µg pcDNA3.1- EVH1-β-geo or 3 µg pcDNA3.1-β-geo) or in approximately twofold excess (3 µg pcDNA3.1- SPRED1 + 7 µg pcDNA3.1-EVH1-β-geo or 7 µg pcDNA3.1-β-geo). In order to induce Ras/ERK/MAPK pathway activation, cells were stimulated with 10ng/ml EGF after transfection. After cell lysis, we examined ERK phosphorylation by Western blot analysis.
Figure 17: Putative Dominant Negative Effect of EVH1-β-geo on Ras/ERK/MAPK Signaling HEK 293 T cells were transfected either with SPRED1 or in combination with EVH1-β-geo or β-geo expression plasmids, stimulated with 10 ng/ml EGF, and cell lysates were immunoblotted with anti-ERK and anti-P-ERK antibodies. SPRED1 was able to inhibit the EGF-induced ERK phosphorylation, while coexpression of EVH1‐β-geo in deficit (3 µg SPRED1 + 3 µg EVH1-β-geo) did not exert any further effect on Ras/ERK/MAPK activity. However, expression of SPRED1 and EVH1-β-geo, the latter in approximately twofold excess (3 µg SPRED1 + 7 µg EVH1-β- geo), revealed an increased ERK phosphorylation in HEK 293 T cells in comparison to cells expressing solely SPRED1. This suggests a possible dominant negative effect of the EVH1-β-geo fusion protein on Ras/ERK/MAPK signaling. Coexpression of sole β-geo without the EVH1 domain had no effect on SPRED1-mediated inhibition of ERK phosphorylation. Unstimulated mock-transfected cells served as negative control.
In comparison to the unstimulated mock-transfected control, stimulation of mock-transfected cells with EGF led to a substantial increase in ERK phosphorylation, which could be considerably inhibited by SPRED1. In cells cotransfected with SPRED1 and EVH1-β-geo in deficit, ERK phosphorylation was reduced comparably to cells transfected only with SPRED1. However, expression of SPRED1 and of EVH1-β-geo in double amount revealed increased 74
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ERK phosphorylation in comparison to cells expressing just SPRED1 or SPRED1 and EVH1-β- geo in deficit. ERK phosphorylation in cells transfected with SPRED1 and β-geo, either in deficit or excess, was comparable to that in cells expressing solely SPRED1 or SPRED1 and EVH1-β- geo in deficit. This analysis showed that β-geo alone has no effect on SPRED1-mediated inhibition of MAPK signaling, while EVH1-β-geo, at least when expressed in excess, seemed to cause an impaired reduction of ERK phosphorylation by SPRED1. Thus, the EVH1 domain in the EVH1-β-geo fusion protein may exert a dominant negative effect on the SPRED1-mediated inhibition of ERK activity in our SPRED2 KO mice, leading to a more active Ras/ERK/MAPK pathway and to an aggravated phenotype.
5.4. Obvious and Versatile Phenotype in SPRED2 KO Mice
From birth on, SPRED2-deficient mice are viable but already significantly smaller as compared to their WT littermates. Hence, we could publish previously that loss of SPRED2 causes dwarfism (Figure 5) and we thereby identified SPRED2 as a novel regulator of chondrocyte differentiation and bone growth ((Bundschu et al., 2005), 3.2.6.1). Till that time I continued the SPRED2 KO mouse project in 2005, no long term observations have been conducted in order to investigate further effects of SPRED2 deficiency in adult mice. However, the breeding and comprehensive investigation of SPRED2 KO mice older than three months showed multiple affections in addition to the dwarf phenotype, such as an increasing deterioration of health state, sudden death due to unknown reasons, and multiple behavioral abnormalities, e.g. excessive drinking and obsessive grooming. To clarify if these phenotypical effects were caused by organ defects, mice at different ages were examined for pathological alterations.
5.5. SPRED2 Deficiency in Mice Leads to Hydronephrosis, Renal Atrophy, and Kidney Deterioration
In fact, dissections of mice older than three months, either died of unknown reasons or sacrificed for tissue sampling, revealed various degrees of renal pathological incidents. Extreme examples were characterized by severely distended kidneys filling large parts of the abdomen 75
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(Figure 18 A). Kidneys were merely composed of a transparent capsule filled with a huge amount of liquid. Simultaneously, progressive atrophy of cortex and medullar zones led to a complete destruction of renal tissue, which clearly reflected a hydronephrosis-like disease pattern. Comparison of whole dissected organs demonstrated that affected kidneys of KO mice appeared pale and not sufficiently supplied with blood (Figure 18 B). In the course of affection, kidneys became deformed due to the fluid-inflated renal pelvis and the developing atrophy. They easily collapsed, especially after artificially induced effluence of liquid. Whereas the left kidney in Figure 18 B was severely damaged by hydronephrosis, which commonly developed unilateral, the right kidney seemed to act compensatory at this stage of disease.
Figure 18: Hydronephrosis and Kidney Damage in SPRED2 KO Mice A) Exterior view of a heavily affected and inflated left kidney, characterized by an extreme accumulation of liquid and a parallel decline of renal cortex and medulla, both common consequences of hydronephrosis. B) As a result of hydronephrosis and renal atrophy, affected isolated and liquid-drained kidneys of SPRED2 KO mice (left KO kidney) appeared flabby, exsanguinous, and morphologically altered. Because the hydronephrosis developed usually unilateral, the contralateral KO kidney (right KO kidney) seemed to act compensatory.
For detailed analysis of renal morphology, paraformaldehyde-fixed WT and KO kidneys were embedded in paraffin, cross- and longitudinal sections were prepared, and stained with H&E. In comparison to the WT kidney (Figure 19 A) showing normal kidney morphology, histological analysis of sections confirmed hydronephrosis in KO kidneys (Figure19 B-D). In particular, strongly dilated renal pelves (Figure 19 B-D, asterisks) and Bowman´s capsules (Figure 19 B-F, arrowheads) were indicative for hydronephrosis-associated fluid retentions. Completely destroyed lateral cortex and mark regions due to massive lymphocytic infiltration (Figure 19 B-F, arrows) further emphasized atrophy of renal tissue in KO kidneys, which is also a common consequence of hydronephrosis.
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Figure 19: Progressive Atrophy and Severe Lymphocytic Infiltration in Kidneys of SPRED2 KO Mice H&E-stained cross sections of A) WT and B) C) SPRED2 KO kidneys and D) H&E-stained longitudinal section of SPRED2 KO kidneys supported the diagnosis of hydronephrosis. Especially KO kidneys displayed extremely dilated renal pelves (asterisks) and Bowman´s capsules (arrowheads) caused by the accumulated liquid. This was accompanied by severe atrophy of cortex and medullar zones and by prominent invasion of lymphocytes (arrows). E) F) Detailed view of strongly inflated Bowman´s capsules (arrowheads) and lymphocytic infiltration (arrows) in SPRED2 KO kidneys. Scale bars: 500 µm.
5.6. Hydronephrosis Is Associated with Tubular Breakdown and Apoptosis in SPRED2 KO Mice
In order to investigate if apoptotic processes contribute to the observed kidney degeneration, we examined kidney morphology on cellular level by H&E-stained paraffin kidney sections. KO kidneys displayed degeneration and atrophy of renal cells, especially in tubular cells (Figure 20 A and B, arrows), whereas glomeruli seemed to remain morphologically intact (asterisks). Since atrophy is a general process of reabsorption and breakdown of tissues and involves apoptosis on a cellular level, we performed TUNEL assays. Apoptotic cells in cryosections of WT and KO kidneys were stained by labeling fragmented DNA at 3´-OH ends with flourescein-12-dUTP using the enzyme terminal deoxynucleotidyl transferase. Compared to WT kidneys (Figure 20 C), the number of TUNEL-positive cells was clearly elevated in KO kidneys, with the majority detected in kidney cortex (Figure 20 D). This was also attested by a higher magnification of WT and KO kidney sections (Figure 20 G and H). To gain an impression of the apoptotic cell rate, cell nuclei were counterstained with DAPI in both overviews (Figure 20 E and F) and magnified 77
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Figure 20: Tubular Breakdown and Apoptosis Contribute to Kidney Degeneration SPRED2 KO Mice H&E-stained paraffin sections of A) WT and B) KO kidneys disclosed the decline of renal tubules (arrows), while glomeruli were unaffected in SPRED2-deficient mice. TUNEL assays with cryosections of C) G) WT and D) H) SPRED2 KO kidneys labeling apoptotic cells by green fluorescence further demonstrated an increase of apoptosis in renal cells of KO kidneys. C) D) Overviews containing kidney cortex and outer medulla regions. G) H) Detailed views of the cortex. E) F) Overviews and I) J) detailed views of DAPI-counterstainings marking cell nuclei in kidney sections of E) I) WT and F) J) KO mice. Scale bars: 50 µm. 78
5. Results views (Figure 20 I and J), confirming that a substantially higher number of cells underwent apoptosis in KO kidneys. Thus, renal atrophy in SPRED2 KO mice seemed to be caused by apoptotic processes especially in the tubules, which might either be causative for or a consequence of hydronephrosis.
5.7. Kidney Damage in SPRED2 KOs Is Accompanied by Polydipsia, Hyperosmolality, and Higher Serum Salt Load
Figure 21: Excessive Water Uptake Correlates with Increased Serum Osmolality and Elevated Serum NaCl A) Daily water consumption was nearly doubled in SPRED2 KO mice (n(WT/KO)=12; ***p<0.001). B) Polydipsia was provoked by a significantly increased serum osmolality (n(WT/KO)=12; **p<0.01). Hyperosmolality was caused by an augmented serum salt load as demonstrated by significantly elevated C) Na+ and D) Cl- levels (n(WT/ KO)≥12; ***p<0.001). Results are expressed as mean ± SD. Figure reprinted from (Ullrich et al., 2011).
Since renal disorders are often accompanied by changes in salt and water homeostasis, we first analyzed the drinking behavior of WT and KO animals aged six to 24 months over a period of one month. Water bottles were weighed each day, and the daily water uptake was noted.
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Calculation of average daily water consumption in relation to body weight (BW) revealed a nearly doubled water uptake in SPRED2 KO mice (114.7 ± 23.3 µl * g BW-1 * 24 h-1 (WT) vs. 200.0 ± 58.4 µl * g BW-1 * 24 h-1 (KO); n(WT/KO)=12; ***p <0.001; Figure 21 A). Enhanced thirst and increased water consumption are physiological consequences of a decreased blood volume and/or an increase in blood osmolality. Determination of blood osmolality via freezing- point depression in serum of WT and KO mice at the average age of ten months revealed that polydipsia correlated with a significantly elevated serum osmolality in SPRED2 KO mice (310.3 ± 7.8 mosmol/kg (WT) vs. 318.3 ± 4.6 mosmol/kg (KO); n(WT/KO)≥10; **p <0.01; Figure 21 B). In order to elucidate hyperosmolality, we measured the levels of various osmolytes in the same serum samples of WT and KO mice used for the determination of hyperosmolality. Except for increased Na+ (146.3 ± 2.3 mmol/l (WT) vs. 152.1 ± 4.2 mmol/l (KO); n(WT/KO)≥10; ***p <0.001; Figure 21 C) and Cl- (114.4 ± 1.6 mmol/l (WT) vs.120.7 ± 4.1 mmol/l (KO); n(WT/KO)≥10; ***p <0.001; Figure 21 D), the levels of all other measured osmolytes were unchanged or reduced (Table 1). Therefore, in our SPRED2 KO mice the elevated bold salt load seems to be causative for hyperosmolality, which further results in excessive drinking.
5.8. Serum Parameter Analyses Underline Kidney Damage and Indicate SPRED2 Deficiency‐Provoked Homeostatic Imbalance
In order to further examine the kidney phenotype and the generally bad state of health of our SPRED2 KO mice, we determined in addition to salt and osmolality parameters a variety of common routine serum parameters in WT and KO mice aged ten months at average (Table 1, survey of analyzed parameters). + - 2+ 3- + Within the measured ion parameters Na , Cl , Ca , PO4 , and K , no significant changes could be detected apart from Na+ and Cl- (Figure 21 C and D, Table 1). However, K+ levels (5.2 ± 1.1 mmol/l (WT) vs. 4.9 ± 0.5 mmol/l (KO); n(WT/KO)≥10; n.s.) were obviously but not significantly decreased in the KOs. Together with the observed hypernatremia, we considered this as a possible indication for hyperaldosteronism. While the kidney parameters creatinin and uric acid were unchanged, urea levels (38.4 ± 11.1 mg/dl (WT) vs. 51.9 ± 26.3 mg/dl (KO); (WT/KO)≥10; n.s.) were apparently but not significantly increased in KOs, corroborating a functional renal disorder, a post-renal blockage of urine flow, or both.
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WT KO
Parameter
Osmolality (mosmol/kg) 310.3 ± 7.8 318.3 ± 4.6** Na+ (mmol/l) 146.3 ± 2.3 152.1 ± 4.2*** Cl- (mmol/l) 114.4 ± 1.6 120.7 ± 4.1*** K+ (mmol/l) 5.2 ± 1.1 4.9 ± 0.5 Ca+ (mmol/l) 2.2 ± 0.1 2.2 ± 0.2 3- PO4 (mmol/l) 2.4 ± 0.2 2.4 ± 0.4 Urea (mg/dl) 38.4 ± 11.1 51.9 ± 26.3 Creatinine (mg/dl) 0.12 ± 0.04 0.10 ± 0.00 Uric Acid (mg/dl) 0.88 ± 0.47 0.81 ± 0.36 Glucose (mg/dl) 216.6 ± 52.7 140.6 ± 28.3*** Triglyceride (mg/dl) 72.8 ± 10.7 51.5 ± 18.3* Cholesterol (mg/dl) 154.4 ± 32.6 109.0 ± 24.1** Alkaline Phosphatase (U/l) 69.4 ± 27.3 48.8 ± 13.3* GOT/AST (U/l) 145.5 ± 71.2 69.2 ± 18.6* GPT/ALT (U/l) 73.1 ± 35.5 34.4 ± 9.4* GGT (U/l) 0.10 ± 0.00 0.10 ± 0.00 Bilirubin (mg/dl) 0.10 ± 0.00 0.12 ± 0.04 Iron (µg/dl) 150.8 ± 25.8 146.9 ± 30.7 Lactat Dehydrogenase (U/l) 363.8 ± 120.9 279.7 ± 179.4 Cholinesterase (U/l) 7985 ± 1178 6354 ± 2239 Total CK (U/l) 636.2 ± 242.3 101.2 ± 42.1** Albumin (g/dl) 2.9 ± 0.3 2.7 ± 0.3 Total Protein (g/dl) 4.9 ± 0.3 4.9 ± 0.7 Amylase (U/l) 2870 ± 522 2954 ± 757 Lipase (U/l) 26.7 ± 12.1 18.7 ± 5.2
Table 1: Examination of Standard Parameters in Serum of WT and SPRED2 KO Mice GOT/AST = Glutamate Oxaloacetate Transaminase/Aspartate Aminotransferase; GPT/ALT = Glutamate Pyruvate Transaminase/Alanine Aminotransferase; GGT = γ-Glutamyltransferase; CK = Creatine Kinase; n(WT/KO) ≥10 for all parameters; *p<0.05; **p<0.01; ***p<0.001; results are expressed as mean SD. Table modified from (Ullrich et al., 2011).
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Based on polydipsia, renal damage, decreased body weight, and adynamia, we considered diabetes mellitus as a possible underlying disease in our SPRED2 KOs. Lack of insulin or a decreased insulin effect leads to an increase in serum glucose and lipolysis in diabetes mellitus patients. However, because of a 35% reduction in serum glucose (216.6 ± 52.7 mg/dl (WT) vs. 140.6 ± 28.3 mg/dl (KO); n(WT/KO)≥10; ***p <0.001) and of a nearly 30% reduction in triglycerides (72.8 ± 10.7 mg/dl (WT) vs. 51.5 ± 18.3 mg/dl (KO); n(WT/KO)≥10; *p <0.05) and in cholesterol (154.4 ± 32.6 mg/dl (WT) vs. 109.0 ± 24.1 mg/dl (KO); n(WT/KO)≥10; **p <0.01), we could exclude diabetes mellitus as reason for the bad health state in the KO mice. In line with the dwarf phenotype of SPRED2-deficient mice (Bundschu et al., 2005), alkaline phosphatase (69.4 ± 27.3 U/l (WT) vs. 48.8 ± 23.3 U/l (KO); n(WT/KO)≥10; *p <0.05) was decreased by 30%, which is indicative for bone diseases, hereunder achondroplasia. The liver parameters GOT/AST (145.5 U/l ± 71.2 (WT) vs. 69.2 ± 18.6 U/l (KO); n(WT/KO)≥10; *p <0.05) and GPT/ALT (73.1 ± 35.5 U/l (WT) vs. 34.4 ± 9.4 U/l (KO); n(WT/KO)≥10; *p <0.05), when elevated very important and specific indicators for severe liver diseases, were dramatically reduced in SPRED2 KO mice. However, a diagnostic relevance of a decrease in these parameters is not known. Liver-specific GGT levels were unchanged, as well as the liver- associated parameters bilirubin, iron, lactate dehydrogenase, and cholinesterase. While level changes of bilirubin, iron, and lactate dehydrogenase besides liver function can further affect blood cell formation, changes in lactate dehydrogenase and cholinesterase can also be indicative for heart and skeletal muscle diseases. A very specific indicator for heart muscle damage is the elevation of total CK. H owever, this parameter was substantially decreased in the SPRED2 KOs (636.2 ± 242.3 U/l (WT) vs. 101.2 ± 42.1 U/l (KO); n(WT/KO)≥10; **p <0.01). Again, no physiological relevance has been assigned to a CK reduction. At least, the levels for albumin and total protein, general parameters of metabolism and nutrition, and amylase and lipase, when upregulated clear indicators for pancreatitis, did not differ between WT and SPRED2 KO mice. Taken together, this routine serum parameter analysis of WT and SPRED2 KO mice clearly reflected a water-electrolyte imbalance associated with kidney dysfunction. Furthermore, it mirrored the recently published hypochondroplasia-like bone disorder. The decrease in numerous of the estimated metabolites (e.g. glucose, triglycerides, cholesterol) and enzyme levels (e.g. GOT/AST, GPT/ALT, total CK) suggested a disorder in body homeostasis and energy balance of SPRED2 KO mice.
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5.9. Loss of SPRED2 Leads to Dysregulation of Salt and Water Homeostasis‐Controlling Hormones
5.9.1. Augmented Aldosterone Secretion Is Accompanied by Elevated Expression of Aldosterone‐Synthase
Aldosterone is one potent regulator of fluid and salt homeostasis by stimulating the reabsorption of Na+ and water and the secretion of K+ in the kidneys.To clarify whether hyperosmolality, hypernatremia, and the tendency to hypokalemia in SPRED2 KO mice were associated with an elevated aldosterone release, we estimated aldosterone in serum of WT and KO mice at the average age of eight months by EIA. Indeed, serum aldosterone (365.6 ± 146.9 pg/ml (WT) vs. 600.1 ± 289.2 pg/ml (KO); n(WT/KO)=14; **p <0.01) was almost doubled in SPRED2 KO mice (Figure 22 A).
Figure 22: Increased Adrenal Aldosterone Synthase Expression Elicits Elevated Aldosterone Secretion A) The observed hyperosmolality and hypernatremia in our SPRED2-deficient mice were provoked by almost doubled serum aldosterone levels (n(WT/KO)=14; **p<0.01). B) Immunohistochemistry of aldosterone synthase in adrenal glands of WTs showed the common expression pattern restricted to the zona glomerulosa (arrows). C) In SPRED2 KO mice, the amount of adrenal aldosterone synthase was increased and the site of expression was obviously broadened (arrows). AS = aldosterone synthase; scale bars: 200 µm; results are expressed as mean ± SD. Figure modified from (Ullrich et al., 2011).
The production of aldosterone from 11-deoxycorticosterone is catalyzed by the aldosterone synthase, which is primarily expressed in aldosterone-producing cells in the zona glomerulosa of the adrenal cortex. To clarify if the hyperaldosteronism in SPRED2 KO mice was accompanied by an increase in aldosterone synthase expression, we performed immunohistochemistry with aldosterone synthase-specific antibodies. In WT adrenal glands, aldosterone synthase staining showed its regular expression pattern restricted to the zona 83
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5.9.2. Hyperaldosteronism Develops Independently of Renin‐Angiotensin
System Activity
Aldosterone production and aldosterone synthase expression is mainly controlled by K+ and the RAS since Ang II potently stimulates the secretion of aldosterone from the adrenal cortex. Because K+ was rather decreased in the SPRED2 KO mice, we investigated if elevated aldosterone release was accompanied by overactivation of the RAS. Therefore, we measured Ang II in serum of WT and KO mice at the average age of ten months by EIA. Unexpectedly, serum Ang II concentration (1.17 ± 0.53 ng/ml (WT) vs. 0.49 ± 0.19 ng/ml (KO); n(WT/KO)≥10; **p <0.01) in the KO mice was reduced by over 50% as compared to WT controls (Figure 23). This suggests that increased aldosterone biosynthesis and secretion was not mediated by the RAS in our SPRED2-deficient mice.
Figure 23: Reduced Ang II Release despite Hyperaldosteronism in SPRED2 KO Mice Although Ang II is known as a primary stimulator of aldosterone release, serum levels were only halved in SPRED2-deficient KO mice (n(WT/KO)≥12); **p<0.01) indicating a RAS-independent development of hyperaldosteronism. Results are expressed as mean ± SD. Figure in part reproduced from (Ullrich et al., 2011).
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5.9.3. Water Deprivation Causes Elevated Loss of Body Fluid in SPRED2 KO Mice
Based on hyperaldosteronism, increased blood salt load, and polydipsia (Figure 21), we analyzed the ability for water retention in SPRED2 KO mice. Therefore, we first determined body weights in SPRED2-deficient mice and WT controls at the average age of ten months before and after experimental water deprivation for 48 h (non-water deprived/basal = (B); water deprived = (WD); Table 2). Because loss of SPRED2 causes hypochondroplasia-like dwarfism (Bundschu et al., 2005), the body weight in the KOs was reduced already before water deprivation (36.6 ± 7.1 g (WT (B)) vs. 23.9 ± 4.0 g (KO (B)); n(WT/KO(B))≥10; ***p <0.001). It further decreased after withdrawal of water (32.2 ± 7.0 g (WT (WD)) vs. 19.3 ± 3.7 g (KO (WD)); n(WT/KO(WD))≥10; ***p <0.001; Table 2). However, while water starvation had no significant effect on body weight within the WT group (36.6 ± 7.1 g 8WT(B)) vs. 32.2 ± 7.0 g (WT(WD)); n(WT(B)/(WD))=10; n.s.), it significantly reduced body weight within the KO group (23.9 ± 4.0 g (KO(B)) vs. 19.3 ± 3.7 g (KO(WD)); n(KO(B)/(WD))≥10; **p <0.01). Accordingly, the relative percental body weight reduction was about 12% in WT but with approximately 19% notably higher in SPRED2 KO mice (Table 2).
Basal Water Deprivation
(B) (WD)
Parameter WT KO WT KO Significance
Body Weight 36.6 ± 7.1 23.9 ± 4.0 32.2 ± 7.0 19.3 ± 3.7 A***; B***; D** (BW, g)
BW Reduction 12.02 19.25 (%)
Table 2: Effect of Water Deprivation on Body Weights of WT and SPRED2 KO Mice Body weights of SPRED2 KO mice and corresponding WT littermates were determined before and after 48 h of water deprivation, revealing a loss of body fluid in both WT and SPRED2 KO mice, but an obviously higher body weight reduction in the SPRED2 KOs. A: WT non-deprived vs. KO non-deprived; B: WT deprived vs. KO deprived; C: WT non- deprived vs. WT deprived; D: KO non-deprived vs. KO deprived; for all parameters: n(WT/KO)≥10; *p<0.05; **p<0.01; ***p<0.001; results are expressed as mean SD. Table in part taken from (Ullrich et al., 2011).
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In a further experiment, we additionally determined absolute weights of adrenals, kidneys, and brains, the corresponding organ/body weight ratios, and serum osmolalites. Determinations were performed in both the water-starved group of WT and SPRED2 KO mice after 48 h of water deprivation and in a second untreated group under basal conditions (Table 3). Despite the dwarf phenotype of SPRED2-deficient mice, absolute organ weights of brain and kidney were unaltered comparing WTs and KOs, and water deprivation had no considerable effect, too. Although not significant, absolute weights of adrenals even tended to be higher in dwarf SPRED2 KOs under basal conditions and further increased after water starvation. (Adrenals: 4.16 ± 0.99 mg (WT(B)) vs. 4.36 ± 1.39 mg (KO(B)) vs. 4.57 ± 0.76 mg (WT(WD)) vs. 5.21 ± 0.67 mg (KO(WD)) ;n(WT/KO(B)/(WD))≥10; n.s); Kidneys: 352.3 ± 90.3 mg (WT(B)) vs. 320.7 ± 41.2 mg (KO(B)) vs. 362.7 ± 83.0 mg (WT(WD)) vs. 323.8 ± 81.6 mg (KO(WD)) ;n(WT/KO(B)/(WD))≥10; n.s); Brains: 493.7 ± 26.3 mg (WT(B)) vs. 506.2 ± 36.9 mg (KO(B)) vs. 517.6 ± 29.6 mg (WT(WD)) vs. 514.2 ± 35.5 mg (KO(WD)); ;n(WT/KO(B)/(WD))≥10; n.s). Due to hypochondroplasia-like dwarfism and the reduced body weights in SPRED2 KO mice already under basal conditions, we focused on relative organ weight changes. Both within the water-starved group and within the basal group, all determined organ/body weight ratios were significantly elevated in the KO mice as compared to the WTs. (Basal group: Adrenals: 0.014 ± 0.005% (WT(B)) vs. 0.019 ± 0.006% (KO(B)); n(WT/KO(B))≥10; *p <0.05; Kidneys: 1.1 ± 0.2% (WT(B)) vs. 1.3 ± 0.1% (KO(B)); n(WT/KO(B))≥10; **p <0.01; Brains: 1.6 ± 0.4% (WT(B)) vs. 2.1 ± 0.2% (KO(B)); n(WT/KO(B))≥10; **p <0.01; Water deprived group: Adrenals: 0.016 ± 0.006% (WT(WD)) vs. 0.028 ± 0.007% (KO(WD)); n(WT/KO(WD))≥10; **p <0.01; Kidneys: 1.2 ± 0.3% (WT(WD)) vs. 1.7 ± 0.2% (KO(WD)); n(WT/KO(WD))≥10; ***p <0.001; Brains: 1.7 ± 0.3% (WT(WD)) vs. 2.7 ± 0.5% (KO(WD)); n(WT/KO(WD))≥10; ***p <0.001). This phenotype-dependent increase in organ weight/body weight ratios could be explained by the hypochondroplasia of SPRED2 KO mice, which is a disproportionate form of dwarfism. Comparing organ/body weight ratios of SPRED2-deficient mice before and after water deprivation, a dramatic increase in ratios was detected for adrenal glands (0.019 ± 0.006% (KO(B)) vs. 0.028 ± 0.007% (KO(WD)); n(KO(B)/(WD))≥13; ***p <0.001), kidneys (1.3 ± 0.1% (KO(B)) vs. 1.7 ± 0.2% (KO(WD)); n(KO(B)/(WD))≥13; ***p <0.001), and brains (2.1 ± 0.2% (KO(B)) vs. 2.7 ± 0.5% (KO(WD)); n(KO(B)/(WD))≥13; ***p <0.001. Contrarily, within the WT group again no significant changes in organ/body weight ratios could be seen (Adrenals: 0.014 ± 0.005% (WT(B)) vs. 0.016 ± 0.006% (WT(WD)); n(WT(B)/(WD))=10; n.s.; Kidneys: 1.1 ± 0.2% (WT(B)) vs. 1.2 ± 0.3% (WT(WD)); n(WT(B)/(WD))=10; n.s.; Brains: 1.6 ± 0.4% (WT(B)) vs. 1.7 ± 0.3% (WT(WD)); n(WT(B)/(WD))=10; n.s.). Correspondingly, in WT mice, water deprivation led to an organ/body weight ratio increase of nearly 15% in adrenals, 9% in kidneys, and 6% in brains. However, in SPRED2 KO mice, in which all organ/body weight ratios were significantly elevated after water starvation, withdrawal
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Basal (B) Water Deprivation (WD)
Parameter WT KO WT KO Significance
Body Weight (BW, g) 32.1 ± 8.4 24.3 ± 2.1 32.2 ± 7.0 19.3 ± 3.7 A**; B***; D*** Adrenal Gland Weight (mg) 4.16 ± 0.99 4.63 ± 1.39 4.57 ± 0.76 5.21 ± 0.67 Adrenal Gland Weight/BW Ratio (%) 0.014 ± 0.005 0.019 ± 0.006 0.016 ± 0.006 0.028 ± 0.007 A*; B**; D*** Adrenal Gland Weight/BW Ratio Increase (%) 14.29 47.37 Kidney Weight (mg) 353.2 ± 90.3 320.7 ± 41.2 362.7 ± 83.0 323.8 ± 81.6 Kidney Weight/BW Ratio (%) 1.1 ± 0.2 1.3 ± 0.1 1.2 ± 0.3 1.7 ± 0.2 A**; B***; D*** Kidney Weight/BW Ratio Increase (%) 9.09 30.77 Brain Weight (mg) 493.7 ± 26.3 506.2 ± 36.9 517.6 ± 29.6 514.2 ± 35.5 Brain Weight/BW Ratio (%) 1.6 ± 0.4 2.1 ± 0.2 1.7 ± 0.3 2.7 ± 0.5 A**; B***; D*** Brain Weight/BW Ratio Increase (%) 6.25 28.57 Serum Osmolality (mosmol/kg) 310.3 ± 7.8 318.3 ± 4.6 331.8 ± 6.2 357.6 ± 23.3 A**; B***; C***; D*** Serum Osmolality Increase (%) 6.93 12.35
Table 3: Effect of Water Starvation on Adrenal, Kidney, and Brain Weights, Corresponding Organ/Weight Ratios, and Serum Osmolalities of WT and SPRED2 KOs Indicated organ weights, organ/body weight ratios, and serum osmolalities of SPRED2 KO mice and corresponding WT littermates were determined under basal conditions in one group of untreated mice and in a second water-starved mouse group after 48 h of water deprivation. While absolute organs weights did not differ significantly between the groups, both organ/body weight ratios and serum osmolalities showed an augmented increase in SPRED2 KOs in comparison to the WTs after water starvation, again indicating water imbalances and fluid loss especially in the KOs. A: WT non-deprived vs. KO non-deprived; B: WT deprived vs. KO deprived; C: WT non-deprived vs. WT deprived; D: KO non- deprived vs. KO deprived; for all parameters: n(WT/KO)≥10; *p<0.05; **p<0.01; ***p<0.001; results are expressed as mean SD. Table in part adopted from (Ullrich et al., 2011).
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5. Results of water led to an organ/body weight ratio augmentation by overall 47% in adrenals, by 31% in kidneys, and by 29% in brains of SPRED2-deficient mice. Thus, the increase in organ/body weight ratios in SPRED2 KOs still rose by 20% in kidney/body weight and brain/body weight ratios, respectively, and even by over 30% in adrenal/body weight ratio. As already demonstrated (see Figure 21 B and Table 1), serum osmolality of SPRED2 KO mice was significantly elevated as compared to WT controls even under basal conditions (310.3 ± 7.8 mosmol/kg (WT(B)) vs. 318.3 ± 4.6 mosmol/kg (KO(B)); n(WT/KO(B))≥10; **p <0.01). Osmolality further increased after water starvation (331.8 ± 6.2 mosmol/kg (WT(WD)) vs. 357.6 vs. ± 23.3 mosmol/kg (KO(WD)); n(WT/KO(WD))≥10; ***p <0.001). Although the increase of serum osmolality after water deprivation was significant within WT (310.3 ± 7.8 mosmol/kg WT(B) vs. 331.8 ± 6.2 mosmol/kg WT(WD); n(WT(B)/(WD))≥10; ***p <0.001) and KO mice (331.8 ± 6.2 mosmol/kg KO(B) vs. 357.6 vs. ± 23.3 mosmol/kg KO(WD); n(KO(B)/(WD))≥13; ***p <0.001), the elevation was with 12% almost doubled in SPRED2 KO mice as compared to a 7% increase in WT animals. Taken together, the water deprivation experiment clearly demonstrated impaired water retention and fluid loss in our SPRED2 KO mice, leading to a stronger decreased body weight, to more elevated organ/body weight ratios, and to a further increased serum osmolality after dehydration as compared to WT littermates.
5.9.4. Vasopressin Response Is Unaffected by Ablation of SPRED2
Figure 24: SPRED2 Deficiency Has no Influence on AVP Release after Water Deprivation Despite basal polydipsia and the extreme loss of body fluid induced by water starvation, no significant differences in serum AVP could be detected after water deprivation (n(WT/KO)≥17; n.s.= not significant). Results are expressed as mean ± SD. Figure in part reprinted from (Ullrich et al., 2011).
Vasopressin is a multifunctional effector in the body, and one of its most important functions is the stimulation of water retention in the kidney. Upon serum hyperosmolality or hypovolemia, AVP induces the integration of aquaporin channels into the apical membrane of renal distal convoluted tubule and collecting duct cells. We wondered if the reduced ability of fluid retention after water deprivation in our SPRED2-deficient mice was caused by an AVP release disorder.
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Therefore, we analyzed AVP levels in serum of WT and KO mice at the average age of ten months by EIA after 48 h of water starvation. Thirst and water deprivation have been shown to significantly increase serum vasopressin. However, unexpectedly, we could not detect any differences between WT and KO AVP levels after water deprivation (79.4 ± 40.7 ng/ml (WT) vs. 74.9 ± 44.9 ng/ml (KO); n(WT/KO)≥10; n.s.; Figure 24). Thus, we assumed that a dysregulation of AVP secretion might not be reasonable for polydipsia in our SPRED2 KO mice.
5.10. Increased Release of the Stress Hormones CRH, ACTH, and Corticosterone from the Hypothalamic‐Pituitary‐Adrenal Axis in SPRED2 KO Mice
In addition to the RAS and K+, ACTH is another regulator of aldosterone release by generally stimulating steroidogenesis in the adrenal gland. Because of the reduced Ang II and K+ levels, excluding an RAS- and K+-dependent elevated alsosterone release, we investigated if ACTH might be involved in the development of hyperaldosteronism in SPRED2 KO mice. The production and release of ACTH was measured by EIA using serum samples of WT and KO mice at the average age of ten months. Indeed, we found serum ACTH elevated about 30% (0.90 ± 0,27 ng/ml (WT) vs. 1.27 ± 0,78 ng/ml (KO); n(WT/KO)≥17; *p <0.05; Figure 25 B).
Figure 25: Increased Pituitary ACTH Release in SPRED2 KOs Is Stimulated by Augmented Hypothalamic CRH Secretion and Followed by Elevated Corticosterone Production Upregulation of stress hormone secretion at all levels of the HPA axis in SPRED2-deficient mice was demonstrated by A) a 30% increase in CRH in PVN region-containing brain lysates (n(WT/KO)=20; p***<0.001), B) a 30% increase in serum ACTH (n(WT/KO)≥22; *p<0.05), and C) a more than doubled serum corticosterone level (n(WT/KO)=12; p***<0.001). Results are expressed as mean ± SD. Figure in part reproduced from (Ullrich et al., 2011).
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Together with CRH and corticosterone, ACTH is produced by tissues of the HPA axis and is, besides its function as a regulator of steroidogenesis, also a potent mediator of stress responses. Triggered by the limbic system, CRH is secreted by the PVN of the hypothalamus in response to stress. CRH stimulates the release of ACTH from corticotrope cells of the anterior pituitary into the systemic circulation. In turn, ACTH further induces the release of corticosterone from the zona fasciculata of the adrenal cortex. In order to investigate if the increased ACTH secretion is accompanied by consecutive upregulation also of the upstream acting CRH and the downstream corticosterone, we also determined CRH by ELISA and corticosterone by EIA. CRH released from hypothalamus and exerting its main action in the pituitary was measured in PVN region-containing brain lysates. Corticosterone, like ACTH released into the systemic circulation, was measured from serum samples of WT and KO mice aged ten months at average. With this analysis, we found CRH, the primary ACTH secretagogue, in line with ACTH significantly increased about 30% (499.0 ± 84.6 pg/mg protein (WT) vs. 652.6 ± 98.6 pg/mg protein (KO); n(WT/KO)=20; ***p <0.001) in brain lysates of SPRED2-deficient animals (Figure 25 A). Consistently, also corticosterone, released downstream of ACTH stimulation, was more than doubled in serum of SPRED2 KO mice (315.9 ± 60.3 ng/ml (WT) vs. 635.2 ± 242.0 ng/ml (KO); n(WT/KO)=12; ***p <0.001; Figure 25 C). Thus, we could show a clear upregulation of stress hormone release from all levels of the HPA axis.
5.11. Augmented Hypothalamic‐Pituitary‐Adrenal Stress Hormone Release Is Accompanied by Elevated Hypothalamic‐ Pituitary Growth Hormone Secretion in SPRED2 KO Mice
Bone development and bone growth can be regulated by a variety of signaling pathways, hereunder by FGF/FGFR3/MAPK/ERK signaling and by growth hormone (GH), which is released from the hypothalamic-pituitary-growth axis. Similar to ACTH from the HPA axis, GH is synthesized and stored in the anterior pituitary and secreted by somatotropic cells into the systemic circulation. GH release is stimulated by its liberin growth-hormone-releasing hormone (GHRH). GHRH is, similar to CRH, released from neurosecretory neurons of the hypothalamus, which are located in the arcuate nucleus. GH stimulates the production of insulin-like growth factor 1 (IGF-1) in the liver, which is comparable with ACTH-induced steroidogenesis in adrenal gland. Both GH itself and IGF-1 are able to regulate long bone growth by stimulating chondrocyte proliferation (Isaksson et al., 1982; Isgaard et al., 1988). 90
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Because of the elevated stress hormone secretion from the HPA axis, and because SPRED2 deficiency leads to hypochondroplasia-like dwarfism (Bundschu et al., 2005), we wondered if hypothalamic-pituitary-growth hormone release was also dysregulated in the SPRED2 KO mice. We therefore analyzed levels of GH and IGF-1 by ELISA using serum samples of WT and KO mice at an average age of six months. We found both serum GH (2.9 ± 2.7 ng/ml (WT) vs. 8.1 ± 5.2 ng/ml (KO); n(WT/KO)≥15; **p <0.01; Figure 26 A) and IGF-1 (381.6 ± 104.9 ng/ml (WT) vs. 604.9 ± 212.1 ng/ml (KO); n(WT/KO)≥15; ***p <0.001; Figure 26 B) elevated in SPRED2- deficient mice. This finding was on the one hand consistent with a generalized upregulation of hypothalamic- pituitary hormone production; on the other hand it was in stark contrast to the dwarf phenotype of our SPRED2 KO mice.
Figure 26: Elevated Growth Hormone Secretion from the Hypothalamic-Pituitary Axis in SPRED2 KO Mice A) Augmented GH release from the pituitary, which was almost tripled in SPRED2 KOs n(WT/KO)≥15; **p <0.01), elicited B) a subsequent 60% increase in IGF-1 secretion from the liver (n(WT/KO)≥15; ***p <0.001). Results are expressed as mean ± SD. Figure based on (Ullrich et al., 2011).
5.12. Overshooting Stress Hormone Release Is Associated with Obsessive Grooming and Self‐Inflicted Skin Lesions in SPRED2 KO Mice
The HPA axis is a key mediator of stress and responds to neural inputs from the central and peripheral nervous system. Upregulation of HPA hormone secretion has been shown to be involved in a variety of stress-associated and mood disorders like depression, burnout syndrome, or bipolar, borderline, and compulsive disorders.
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Very strikingly, SPRED2 KO mice older than four months developed severe skin lesions. They were usually first noticed as small wounds at the snout or cheek but evolved into uni- or bilateral lesions encompassing large parts of the neck or face (Figure 27). These lesions occurred in SPRED2 KO mice, regardless of whether they were housed individually or together with cage mates. No lesions were observed in WT littermates, even when kept in the same cage together with KO mice. SPRED2-deficient mice were never found behaving aggressively or being busy in grooming other cage mates. However, they were very often and very intensely seen engaged in self-grooming regardless if housed individually or in groups. Long-term observations and video monitoring of SPRED2 KO mice and WT littermates demonstrated that KOs spent clearly more time for grooming as WT controls. SPRED2-deficient mice performed excessive and injurious levels of self-grooming bouts regardless of day or night time, resulting in bloody wounds and purulent lesions. The observed phenotype of our SPRED2 KO mice is reminiscent of a compulsive disorder, which affects regular grooming behavior and manifests as obsessive grooming. It is very likely a consequence of the excessive stress hormone release from the HPA system.
Figure 27: Obsessive Grooming of SPRED2 KO Mice Results in Self-Inflicted Skin Lesions Mice older than four months developed oozing and bloody uni- or bilateral skin lesions, which resulted from excessive self-grooming. In combination with the upregulated stress hormone release from the HPA axis, this behavior might be indicative for an obsessive-compulsive disorder. Figure in part reprinted from (Ullrich et al., 2011). 92
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5.13. SPRED2 Expression Profiling in Hypothalamic‐Pituitary‐ Adrenal Tissues and Kidney
SPRED2 expression has been shown to be almost ubiquitous in mouse tissues as demonstrated previously on RNA and protein levels and by Spred2 promoter activity assays (Bundschu et al., 2006a; Engelhardt et al., 2004). In order to monitor SPRED2 expression in organs involved in the phenotypical pathologies of SPRED2-deficient mice, we especially focused on tissues of the HPA axis and the kidney. We visualized on the one hand Spred2 promoter activity by X-Gal-stainings using whole organs and tissue sections of HET mice; on the other hand, we analyzed the expression pattern of SPRED2 by immunohistochemistry using tissue sections of WT mice and a SPRED2-specific antibody for protein detection. As demonstrated previously, both Spred2 promoter activity and SPRED2 expression is very high in brain. Accordingly, the entire HET brain was blue colored upon X-Gal staining in comparison to the WT control (Figure 28 A). In particular in the hypothalamus, where CRH and AVP are synthesized in the PVN, the Spred2 promoter was clearly active. Hypothalamic Spred2 promoter activity was further confirmed by X-Gal stained brain sections of HET mice, demonstrating an evenly distributed blue staining in the entire organ (Figure 28 B). SPRED2 protein expression was comparable to endogenous promoter activity and also evenly distributed throughout the entire hypothalamus as shown by anti-SPRED2 staining of WT brain sections (Figure 28 C). ACTH is released into the systemic circulation from the anterior, and AVP from the posterior part of the pituitary gland. Spred2 promoter activity was also present in the whole organ (Figure 28 D), which was confirmed by tissue sections (Figure 28 E). SPRED2 protein expression was again detected throughout the whole pituitary but seemed to be especially high in the posterior tract (Figure 28 F). The adrenal gland releases aldosterone from the zona glomerulosa and corticosterone from the zona fasciculata. X-Gal staining of adrenals as well indicated a strong endogenous Spred2 promoter activity (Figure 28 G), which was further defined by sectional analysis. Predominantly a thin layer at the border of the adrenal cortex representing the zona glomerulosa was positive for both promoter activity and SPRED2 expression (Figure 28 H and I), indicating a role of SPRED2 especially in mineralocorticoid synthesis and/or release. Finally, we could demonstrate Spred2 promoter activity and SPRED2 expression in the kidney and thereby confirm our former data. The endogenous Spred2 promoter activity was substantially higher in cortex and outer medulla as compared to inner medulla and papilla (Figure 28 J and K). Blue staining marked especially the zone in kidney cortex, where proximal 93
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Figure 28: Spred2 Expression Profiling in Tissues of the HPA Axis and in Kidney Endogenous Spred2 promoter activity of A) D) G) J) whole WT and HET organs (arrows) and B) E) H) K) tissue sections of SPRED2 HET mice was demonstrated by X-Gal stainings. C) F) I) L) SPRED2 protein expression was shown by immunohistochemistry with tissue sections of WT mice. A) B) C) Both Spred2 promoter activity and SPRED2 expression were evenly distributed throughout the whole hypothalamus. D) E) F) Endogenous promoter activity was detected in the entire pituitary gland, but protein content seemed to be most pronounced in the posterior part. G) H) I) SPRED2 expression and Spred2 promoter activity were especially high in the zona glomerulosa of the adrenal gland, which is the site of aldosterone release. Proximal and/or distal tubules but not the glomeruli were the sites of highest Spred2 promoter activity and protein content in the kidney. J) K) L) Scale bars: B, E, H, K: 500 µm; C, F, I, L: 50 µm. Figure modified from (Ullrich et al., 2011).
and distal tubules are located. A tubules-restricted Spred2 promoter activity was also indicated by the lack of blue staining in renal glomeruli. In line, substantial SPRED2 expression examined
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5. Results by immunohistochemistry was also seen in cortex and in the cortico-medullary boundary, again showing intensely stained renal tubules, while glomeruli were devoid of SPRED2 (Figure 28 L). Taken together, expression profiling verified both profound endogenous Spred2 promoter activity and SPRED2 expression in the tissues of the HPA axis and in the kidney.
5.14. SPRED2 Inhibits Hypothalamic CRH Production and Release by Downregulating CRH Promoter Activity in an ERK/MAPK/Ets1‐Dependent Manner
5.14.1. Increased in vivo ERK Phosphorylation in Brains of SPRED2 KOs
A very important question we still had to address was the mechanism how SPRED2 could be involved in the dysregulation of the HPA axis. Because SPREDs have been shown to be negative regulators of exclusively Ras/ERK/MAPK signaling (King et al., 2005; Nonami et al., 2004; Wakioka et al., 2001), we first examined ERK expression and phosphorylation in six and twelve months old WT and SPRED2 KO animals. Since the hypothalamus is part of the limbic system, the site of CRH release from parvocellular neurons of the PVN, and thus the most upstream trigger of the HPA axis, we used the same brain lysates as for CRH ELISA because they mainly contained the hypothalamic PVN. ERK/MAPK activity in these brain lysates was investigated by Western blot analysis using anti-ERK and anti-P-ERK antibodies. In both six and twelve months old animals, we detected an obviously increased ERK phosphorylation in the SPRED2 KOs as compared to WTs, while the unphosphorylated form of ERK was expressed equally (Figure 29 A). ERK phosphorylation was quantified by determination of signal strengths of ERK and P-ERK and expressing them as arbitrary units. For each sample, P-ERK signals were normalized to the corresponding ERK signals, i.e. the ratio of P-ERK to ERK was calculated and the mean of the WT ratios was set as 1. This quantification revealed an over 2.5-fold elevated P-ERK to ERK ratio in both age groups of SPRED2 KO mice as compared to the WT groups (Figure 29 B; 6 months old mice: 1 ± 0.35 arbitrary units (WT) vs. 2.73 ± 0.73 arbitrary units (KO); n(WT/KO)≥8; ***p<0.001; 12 months old mice: 1 ± 0.56 arbitrary units (WT) vs. 2.58 ± 0.42 arbitrary units (KO), n(WT/KO)≥7; ***p<0.001). Due to the loss of SPRED2-mediated inhibition, ERK activation was upregulated in the PVN- containing hypothalamic region of SPRED2 KOs. Thus, we could confirm the suppressive effect of SPRED2 on Ras/ERK/MAPK signaling and demonstrate it initially in vivo in mouse brain. 95
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Figure 29: Increased Ras/ERK/MAPK Signaling in PVN-Containing Brain Regions of SPRED2-Deficent Mice A) Western blot analysis with anti-ERK and anti-P-ERK antibodies using hypothalamus lysates of both six and twelve months old SPRED2 KOs revealed augmented ERK phosphorylation in both young and aged mice in comparison to WT controls. B) Quantification of ERK and P-ERK signals demonstrated a more than 2.5-fold increase of the P- ERK/ERK ratio both in young and aged SPRED2-deficient mice (n(WT/KO)≥7); ***p<0.001). Results are expressed as mean ± SD. Figure in part reprinted from (Ullrich et al., 2011).
5.14.2. SPREDs Suppress CRH Transcription in vitro
Because SPRED2 downregulates MAPK signaling and ERK phosphorylation in the hypothalamus, we aimed to investigate if SPRED-mediated Ras/ERK/MAPK pathway regulation also has a direct impact on hypothalamic CRH production. To date, the only known functional cellular role of SPREDs is the exclusive inhibition of Ras/ERK/MAPK signaling, one of its main consequences is transcriptional regulation of target genes. Thus, we assumed that the most likely way how SPRED could influence CRH release is by regulating CRH transcription. To be able to monitor possible SPRED effects on CRH transcription in vitro, we used a reporter vector containing a firefly luciferase reporter gene expressed under control of the CRH promoter. To generate this CRH promoter reporter we amplified a 5.7 kb region of the 5´ CRH promoter sequence including exon 1 by PCR from genomic mouse DNA and cloned it into the firefly luciferase reporter vector pGL3 (Appendix 9.3.). Due to the occurrence of RE-1/NRSE, a critical regulatory element in intron 1 of the CRH gene, which is able to silence CRH transcription (Seth and Majzoub, 2001), we cloned both the CRH promoter region with
(CRHProm-RE) and without (CRHProm) the RE-1/NRSE repressor. The embryonic mouse hypothalamus cell line mHypoE-44 was transiently transfected with the CRH promoter firefly luciferase reporters and with an internal control vector containing the Renilla luciferase reporter
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1/NRSE between exon 1 and 2 (pGL3-CRHProm-RE) revealed no luciferase signal in mHypoE-44 cells, which underlined the repressive effect of RE-1/NRSE on CRH transcription (Figure 30 A). Thus, further reporter studies were conducted with the CRH promoter reporter construct lacking the RE-1/NRSE repressor (pGL3-CRHProm, Figure 30 B). mHypoE-44 cells were transfected with the CRH promoter firefly luciferase reporter pGL3-CRHProm, with the Renilla luciferase control vector, with SPRED1 and SPRED2, respectively, either with EVH1-β-geo in approximately trifold excess or without (Figure 30 C). Because CRH is released in response to stress in vivo, mHypoE-44 cells were normally cultured in high glucose and FBS-containing medium but were starved in low glucose medium without FBS for 24 - 30 h after transfection. Firefly and Renilla luciferase reporter activity were determined, luminescent signals of firefly luciferase activity were normalized to Renilla luciferase activity for all samples, and mean relative activity of cells expressing solely the CRH promoter reporter was set to 1.
Figure 30: SPRED1 and SPRED2 Downregulate CRH Promoter Activity in vitro A) Schematic depiction of the cloned CRH promoter luciferase reporter pGL3-CRHProm-RE including the RE-1/NRSE repressor. Approximate positions of the predicted 4 Ets1 binding sites (Ets1), exon 1 (Ex1), the RE-1/NRSE repressive element (RE), and the firefly luciferase reporter gene (Luc) are indicated. B) Schematic depiction of the cloned CRH promoter luciferase reporter pGL3-CRHProm lacking the RE-1/NRSE repressor. Approximate positions of the predicted 4 Ets1 binding sites, exon 1, and the firefly luciferase reporter gene are indicated. C) CRH promoter reporter assay in mHypoE-44 cells using pGL3-CRHProm, which comprises the CRH promoter region without the RE- 1/NRSE repressive element. Both SPRED1 and SPRED2 were able to reduce firefly luciferase reporter gene expression and thus CRH promoter activity to 60%. No further effect on CRH promoter activity was detected by coexpression of EVH1-β-geo in about trifold excess (n=12; ***p<0.001; n.s.=not significant). Results are expressed as mean ± SD. Figure modified from (Ullrich et al., 2011).
These assays revealed that both SPRED1 and SPRED2 were able to suppress the relative luciferase activity of the CRH promoter reporter by about 40% (1 ± 0.18 arbitrary units (CRHProm)
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5. Results vs. 0.61 ± 0.14 arbitrary units (SPRED1) vs. 0.58 ± 0.08 arbitrary units (SPRED1/EVH1-β-geo) vs. 0.56 ± 0.10 arbitrary units (SPRED2) vs. 0.56 ± 0.11 arbitrary units (SPRED2/EVH1-β-geo); n≥12; ***p<0.001). Cotransfection of EVH1-β-geo in excess did not exert any further effect on CRH promoter activity (Figure 30 C). Hence, SPRED1 and SPRED2 can downregulate CRH transcription and expression explaining the elevated CRH production in SPRED2-deficient mice. In contrast to the putative dominant negative effect of EVH1-β-geo on ERK-phosphorylation detected by cotransfection studies in HEK 293 T cells (5.3.3.), the fusion protein seemed to have no effect on CRH promoter activity.
5.14.3. SPREDs Inhibitory Effect on CRH Promoter Activity is Dependent on ERK/MAPK‐mediated Regulation of Ets Transcription Factors
The CRH promoter comprises a manifold set of transcription factor binding sites as predicted by Alibaba 2.1 TF Binding Prediction. Hereunder are a great variety of predicted binding sites for Ets transcription factor family members (Appendix 9.6.). In particular, the cloned CRH promoter reporter region lacking the RE-1/NRSE repressor (CRHProm) contains four binding sites for Ets1, four for PU.1, two for Erg1, and for one Elk1, Elf1, and PEA3 (Figure 30 B). Because transcription factors of the Ets family are favored targets of the Ras/ERK/MAPK cascade, they could provide the link between SPRED-mediated inhibition of Ras/ERK/MAPK signaling and SPRED-mediated suppression of CRH transcription. To investigate if Ets binding sites in the CRH promoter are indeed critical for SPRED-dependent regulation of CRH transcription, we performed an Ets reporter assay. Therefore, we used a plasmid comprising 4 Ets1 transcription factor binding sites, which control the expression of a downstream firefly luciferase reporter gene (Figure 31 A, Appendix 9.5., (Hoffmeyer et al., 1998)). The experimental setup was based on the CRH promoter reporter study, i.e. mHypoE- 44 cells were transfected with the 4xEts1 reporter, with a Renilla luciferase reporter vector as internal control, and with SPRED1 and SPRED2, respectively, either with about trifold amounts of EVH1-β-geo or without. In case of the Ets reporter assay, cells were only starved for FBS but not for glucose for 24 - 30 h after transfection. By this Ets reporter assay, we detected that again both SPRED1 and SPRED2 were able to reduce relative luciferase activity. SPRED expression led to an inhibition of Ets factor-dependent gene transcription by even 90% in mHypoE-44 cells (1 ± 0.47 arbitrary units (Ets1) vs. 0.097 ± 0.044 arbitrary units (SPRED1) vs. 0.071 ± 0.035 arbitrary units (SPRED1/EVH1-β-geo) vs. 0.066 ± 0.031 arbitrary units (SPRED2) vs. 0.055 ± 0.007 arbitrary units (SPRED2/EVH1-β-geo); n≥12; ***p<0.001). The SPRED function on Ets1-mediated transcription was unaffected by coexpression of EVH1-β-geo in excess (Figure 31 B). 98
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Figure 31: SPRED1 and SPRED2 Suppress Ets1- dependent Transcription A) Schematic depiction of the 4xEts1 reporter pGL2- 4xEts1 containing 4 binding sites for the transcription factor Ets1 (Ets1) and the firefly luciferase reporter gene (Luc). B) Both SPRED1 and SPRED2 were able to downregulate Ets1-dependent transcription of the luciferase reporter gene to approximately 10%. EVH1-β- geo coexpression in approximately trifold amounts did not exert any further effect on Ets1-dependent transcriptional activity (n=12; ***p<0.001; n.s.=not significant). Results are expressed as mean ± SD. Figure modified from (Ullrich et al., 2011).
This Ets reporter study again gave no hint on any effect exerted by EVH1-β-geo on Ets-like factor mediated transcription. However, this study confirms that CRH transcription can be regulated by the activation of Ets binding sites, especially Ets1, which are present in the CRH promoter. Thus, it provides an explanation how inhibition of Ras/ERK/MAPK signaling and thereof dependent downregulation of Ets1-mediated transcription by SPREDs can suppress CRH transcription and production.
5.14.4. SPREDs Reduced CRH Release from mHypoE‐44 Cells in vitro
In order to investigate if SPRED-mediated inhibition of CRH transcription also leads to a reduction of CRH release in vitro, we performed a CRH ELISA using the hypothalamic mHypoE- 44 cell line (Figure 32). mHypoE-44 cells were transfected with SPRED1, SPRED2, or empty expression plasmid, starved for glucose and FBS for 36 h, and cell culture supernatants were used for determination of the secreted CRH. Indeed, SPRED1 reduced in vitro CRH secretion by 30% and SPRED2 by 40%, which was in line with the downregulation of CRH transcription (34.2 ± 8.9 pg/ml (Mock) vs. 25.4 ± 3.6 pg/ml (SPRED1) vs. 20.6 ± 2.9 pg/ml (SPRED2); n=9; Mock vs. SPRED1: *p <0.05; Mock vs. SPRED2: **p <0.01). 99
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Figure 32: Reduction of CRH Release from mHypoE-44 Cells by SPRED1 and SPRED2 in vitro ELISAs with cell culture supernatants of mHypoE-44 cells demonstrated a decrease of CRH secretion to 70% by SPRED1 and to 60% by SPRED2 (n=12; *p<0.05 for SPRED1; **p<0.01 for SPRED2). Results are expressed as mean ± SD. Figure in part reprinted from (Ullrich et al., 2011).
5.14.5. Higher Content of CRH mRNA in Hypothalamic PVN‐Containing
Brain Regions of SPRED2‐deficient Mice
In hypothalami of SPRED2 KO mice, we already detected increased ERK phosphorylation (5.14.1.) in combination with augmented CRH release (5.10.) in vivo. The downregulation of CRH promoter activity (5.14.2) mediated by Ets family transcription factors (5.14.3) and of CRH release (5.14.4) by SPREDs has been demonstrated in vitro. In order to ascertain if SPREDs are able to regulate MAPK/ERK-dependent CRH transcription not only in vitro but also in vivo, we quantified the levels of CRH mRNA in hypothalamic PVN regions of WT and SPRED2 KO mice. Therefore, we performed Northern blot analysis with RNA isolated from the PVN- containing region of the hypothalamus and using a part of the mouse CRH cDNA labeled with 32P as a radioactive probe (Figure 33 A, Appendix 9.7.). CRH mRNA levels were quantified by imaging the 32P comprised in the bound probe and by normalizing the signals to the corresponding total RNA content. Both the signal strengths of 32P and total RNA were expressed as arbitrary units, the relative CRH mRNA contents of WT and KO samples were calculated, and WT was set to 1. This quantification revealed a 60% increase of CRH mRNA in the KO mice (Figure 33 B; 1 ± 0.58 arbitrary units (WT) vs. 1.62 ± 0.55 arbitrary units (KO); n(WT/KO)≥8; *p<0.05), indicating a functional role of SPREDs in the regulation of CRH transcription and expression also in vivo. Taken together, this study showed that SPREDs are suppressors of CRH promoter activity, transcription, and release both in vivo and in vitro. SPRED-mediated downregulation of CRH release was accompanied by SPRED-mediated inhibition of ERK/MAPK signaling and a reduced activation of Ets1 factor-dependent transcription, leading to a decreased ERK/MAPK/Ets1-dependent CRH promoter activity. Vice versa, in our SPRED2 KO mice, 100
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ERK/MAPK activity, CRH mRNA levels, and hypothalamic CRH release were accordingly increased. Thus, our study provides a new link how SPRED can mediate the activity of the HPA axis by regulating ERK/MAPK/Ets1 signaling.
Figure 33: Elevated CRH mRNA Levels in Hypothalamic PVN-Containing Brain Regions of SPRED2 KOs A) Northern blot analysis with RNA samples from the PVN-containing hypothalamus of WT and SPRED2 KO mice. Radioactive labeling of CRH mRNA with 32P demonstrated a higher CRH mRNA content in the KOs. B) Quantification of CRH mRNA levels demonstrated a 60% increase in PVN-containing brain regions of SPRED2-deficient mice (n(WT/KO)≥8; *p<0.05). Results are expressed as mean ± SD. Figure modified from (Ullrich et al., 2011).
5.15. Reduced Survival Probability in SPRED2 KO Mice
Most likely associated with the various phenotypical affections and the generally poor health status, our SPRED2 KO mice died obviously earlier as compared to their WT littermates, while in most cases the cause of death was unknown. We determined the survival probability of WT and SPRED2 KO mice by Kaplan-Meier survival analysis. Therefore, we observed 20 SPRED2 KO mice and 20 WT littermate controls over a period of 18 months and documented, if applicable, the age of death. Very strikingly, more than 50% of the SPRED2-deficient KO mice already died within the first five months of age (Figure 34). The outward health was still good in most mice at this age because obsessive grooming, wound development, and kidney degeneration usually begin at the earliest at the age of about four months. However, these comparatively young animals were suddenly found dead in their cages. Further 25% of KO mice died within the remaining 13 months, so that only 25% of KOs survived the whole period of 18 months. In contrast, 80% of all WT mice were still alive at the end of the 18 months (80 ± 8.9% (WT) vs. 25 ± 9.7% (KO); n(WT/KO)=20; ***p<0.001). This results in a 55% reduction of survival probability in SPRED2 KO mice KOs as compared to WTs.
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Figure 34: Kaplan-Meier Survival Analysis of SPRED2 KO Mice and WT Littermate Controls In comparison to WT mice, of which 80% survived the investigated time period of 18 months, only 25% of SPRED2 KOs survived the whole time span, revealing a 55% reduction of survival probability in KOs (n(WT/KO)=20; ***p<0.001).
Hence, we could show a significantly reduced life expectancy for SPRED2 KO mice. The unclear sudden death of especially younger animals does not necessarily have a causal relationship to the phenotypical affections described in this work. It rather points towards paroxysmal disorders that might either emanate from the cardiovascular system, e.g. myocardial infarction or stroke or from the nerve system e.g. epilepsia. Two further studies have already been initiated to investigate the possible causes for the reduced survival probability of our SPRED2 KO mice and to further characterize phenotypical consequences provoked by SPRED2 deficiency: One study to explore the effects of SPRED2 deficiency on the cardiovascular system and another study to further unravel behavior and synaptic function in SPRED2-deficient mice.
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Discussion 6
6.1. The SPRED2 Gene Trap Model – Possible Effects of the EVH1‐β‐geo Fusion Protein
In 2001, SPRED proteins were discovered by a yeast to hybrid screen, they were named after the related Sprouty proteins with whom they share a homologous C-terminus, and they were described as exclusive inhibitors of Ras/ERK/MAPK signaling (Wakioka et al., 2001). At the same time, our group also detected the SPREDs due to their homology to the EVH1 domain of VASP. In order to unravel the completely unknown in vivo function of SPRED proteins, we generated a KO mouse model lacking functional SPRED2 protein expression. For disruption of the Spred2 gene in mice, we used the very fast gene trap method, which allowed a functional knockout of the target gene in combination with gene expression profiling by X-Gal staining (Bundschu et al., 2006a; Bundschu et al., 2005; Ullrich and Schuh, 2009). With this study, we could show and confirm that gene trapping resulted in a loss of full-length SPRED2 in SPRED2-deficient mice, as demonstrated by Western blot. Surprisingly, in WT kidney lysates instead of the common 46 kDa SPRED2 isoform expressed for example in WT brain, we detected the expression of a substantially smaller isoform of about 30 kDa (5.3.1., Figure 15 A). By screening of human expressed sequence tag (EST) databases in an earlier experiment indicating that the Spred2 gene generates at least three forms of transcripts (Engelhardt, 2004), we already got a hint for the appearance of SPRED splice variants. Furthermore Eve-3 has been described as a liver-specific splice variant of SPRED3, thus confirming the natural occurrence of SPRED splice variants (King et al., 2006). The gene trap vector, which disrupts the endogenous Spred2 gene in our mouse model, is inserted between exons 4 and 5. It was repeatedly described that exons upstream of the disruption point were still used as an RNA template regardless if the mouse models were generated by homologous recombination or by gene trapping. This may lead to the expression
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6. Discussion of either a truncated protein or a truncated splice variant or of read-through products containing both exonic and vector information (Bula et al., 2005; Calfa et al., 2011; McClive et al., 1998). Because Spred2 was interrupted downstream of exon 4, the expression of exons 1 - 4 primarily representing the EVH1 domain could not be excluded. Indeed, besides the sole β-geo reporter, we detected the expression of a fusion protein comprising the EVH1 domain and β-geo. This was in contrast to our recent studies, in which Northern and Western blot analysis using RNA or protein lysates prepared from SPRED2 KO brains failed to detect any expression of full-length- SPRED2, any truncated SPRED2, or any enlarged read-through product (Bundschu, 2005). However, in this present study, the detection of the EVH1-β-geo was possible by Western blot not only in brain lysates, one of the tissues with highest natural SPRED2 expression, but also in kidney and both by anti-SPRED2 and anti-β-gal antibodies. One reason for the now possible detection of EVH1-β-geo could be the affinity of the anti-β-gal antibody used exclusively in this study. It seemed to be higher for EVH1-β-geo as the affinity of the anti-SPRED2 antibody used in the former studies. Moreover, the anti-SPRED2 antibody used in the present study was freshly purified from serum of immunized rabbits, might have a better quality, and thus detect also lower amounts of protein. Too low levels of RNA or protein might be a further reason for the undetectable EVH1-β-geo in the former study. In order to investigate possible effects, which EVH1-β-geo might exert on the remaining SPRED1 activity and thus on SPRED mediated Ras/ERK/MAPK signaling in our SPRED2 KO mice, we subcloned EVH1-β-geo and β-geo. Their expression and functionality was confirmed in transfected HEK 293 T cells by Western Blot and X-Gal stainings. Expression of SPRED1 and higher amounts of EVH1-β-geo in EGF-stimulated HEK 293 T cells showed a less reduced ERK phosphorylation in comparison to cells expressing only SPRED1. This obvious increase in ERK phosphorylation mediated by EVH1-β-geo led us to the assumption that the EVH1-β-geo fusion protein might exert a dominant negative effect on Ras/ERK/MAPK signaling. Although both the EVH1 and the SPR domain of SPREDs have been shown to be required for appropriate function, the EVH1 domain rather seems to exert the biochemical inhibitory function on the Ras/ERK/MAPK pathway, while the SPR domain triggers the protein localization on cell membranes and association with interaction partners. The EVH1 domains of Ena/VASP, Homer/Vesl, and WASP protein families are also popular protein interaction modules (Fedorov et al., 1999). However, due to a narrower binding groove, a different mechanism of interaction and a binding to less proline-rich motifs is assumed for the SPRED proteins in comparison to the other EVH1 domain-containing proteins (Harmer et al., 2005; Zimmermann et al., 2004). Maybe associated with this predicted divergent binding modus, to date only two binding partners of the SPRED-EVH1 domain have been identified yet (Mardakheh et al., 2009; Stowe et al., 2012). Strikingly, interaction with both proteins indeed mediate inhibitory functions of SPREDs: Binding of SPRED2 to NBR1 targets the FGFR to lysosomal degradation and binding 104
6. Discussion of SPRED1 to the RasGAP NF1 inactivates Ras by inducing the hydrolysis of active Ras-GTP, both resulting in downregulation of Ras/ERK/MAPK signaling (Mardakheh et al., 2009; Stowe et al., 2012). Thus, in our SPRED2 KO mice, the functional EVH1 domain in the EVH1-β-geo fusion protein might intercept SPRED1 binding partners needed for proper SPRED-mediated Ras/ERK/MAPK pathway inhibition and therefore evoke a less reduced ERK activity. A possible dominant negative effect of the EVH1-β-geo fusion protein is further supported by ∆C-SPRED1 and ∆C-SPRED2 mutants missing the SPR but still containing the EVH1 domain. These mutants also demonstrate dominant negative behavior in different cell types and after stimulation of Ras/ERK/MAPK signaling with different agonists (King et al., 2005; Nonami et al., 2004; Wakioka et al., 2001). Similarly, the EVH1 domain of the related VASP was also shown to act dominant negative when overexpressed in cardiac myocytes (Eigenthaler et al., 2003). In contrast to the possible dominant negative effect of EVH1-β-geo on ERK activity, neither in the CRH promoter reporter assay nor in the Ets transcription factor reporter assay we could detect any effect of EVH1-β-geo on SPRED-mediated transcriptional regulation (5.14.2./5.14.3.) Although we suppose that SPRED-mediated inhibition of Ets factor-dependent CRH transcription is a downstream consequence of SPRED-mediated suppression of Ras/ERK/MAPK signaling, the two assays might not directly be comparable. While the ERK phosphorylation assay was performed in HEK 293 T cells, the transcriptional reporter assays were performed in the hypothalamic mHypoE-44 cell line. Furthermore, Ets-dependent CRH transcription is very likely activated by the Ras/ERK/MAPK cascade but maybe in part also coinfluenced by other pathways, which are not affected by SPREDs. Thus, the effect that EVH1-β-geo might exert on SPRED function might be dependent on cell type, tissue, and regulated process and on targeted and involved factors. An indication that a dominant negative effect of EVH1-β-geo may at least partly play a role in the regulation of physiological processes in our SPRED2 KOs is the very complex and versatile phenotype including dwarfism (Bundschu et al., 2005), HPA axis dysregulation, salt and water imbalances, and cardiac, neuronal, behavioral, and mood disorders (see results 5.9.,5.10., 5.11., and 7.). In contrast, two further existing SPRED1 and SPRED2 KO mouse models have comparably mild phenotypes (Denayer et al., 2008; Inoue et al., 2005; Nobuhisa et al., 2004; Nonami et al., 2004; Phoenix and Temple, 2010). Therefore, the phenotype of SPRED2 KO mice generated in our group resembles most likely that of SPRED2 KO/SPRED1 HET mice but is less severe than that of the embryonic lethal SPRED1/2 double KO mouse model (Nobuhisa et al., 2004; Taniguchi et al., 2007). This may be caused by the additional dominant negative effect of EVH1-β-geo in our SPRED2 KO mice, leading to an impaired inhibitory effect of the remaining SPRED1 on the Ras/ERK/MAPK pathway.
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6.2. Causes of Kidney Damage in SPRED2 KO Mice – An Interplay of Hydronephrosis, Atrophy, and Apoptosis
SPRED2 KO mice older than six months showed various stages of kidney degeneration, which was induced by hydronephrosis. Hydronephrosis is usually caused by obstruction of free urine flow out of the kidney leading to an upward transmission of uretral pressure and thus to distension and dilation of the renal pelvis. The causative obstruction may be partial or complete, unilateral or bilateral, and may develop acutely or chronically throughout the whole urinary system including kidneys, ureters, bladder, and urethra. Hence, hydronephrosis can result from various abnormal pathophysiological occurrences in the urinary tract already developing during fetal development, including congenital structural abnormalities of the junctions between the kidney, ureter, and bladder. Non-congenital and non-structural causes in adulthood leading to obstructions of the urinary tract comprise urinary stones, blood clots, retroperitoneal fibrosis within the urinary system, or external pressure on the urinary system e.g. by tumors or abnormally placed blood vessels. At last, hydronephrosis can also result from reflux of urine from the bladder back into the kidneys, which could be a consequence of prostatic enlargement, abnormal bladder muscle contraction, or abdominal tumors. Blocking of urine outflow or reflux will commonly result in infections and inflammatory reactions of the urinary tract, further leading to progressive renal atrophy and kidney failure. Indeed, in our SPRED2 KO mice, hydronephrosis was accompanied by considerable atrophy of renal tissue and increased apoptosis of renal tubules. Characteristically, we observed mostly unilateral hydronephrosis associated with dramatic pelvis dilatations, parenchymal thinning, and loss of normal renal morphology. Affected and dilated kidneys further showed lymphocytic infiltration, which was indicative for inflammatory and infectious processes. In contrast, the contralateral kidneys were structurally and functionally still intact and seemed to compensate renal function. Apoptosis, as also detected in kidneys of our SPRED2 KO mice, is considered to be the main process of cell death in renal parenchymal cell demise and atrophy and is often involved in the initiation and progression of a variety of renal disorders (Amore and Coppo, 2000; Ortiz, 2000). In our SPRED2 KO mouse model, hydronephrosis occurs in parallel to renal atrophy and apoptosis. However, the causal relationship, i.e. if apoptosis of kidney cells occurs first and may be causative for hydronephrosis and renal atrophy or, vice versa, if the occurrence of hydronephrosis may lead to apoptosis and renal atrophy remains to be elucidated. Because kidney cells are armed with the genetic machinery to undergo apoptosis, this process is likely to play a physiological role in the kidney. In mature and healthy kidneys, an equilibrium 106
6. Discussion between cell proliferation and apoptosis keeps the turnover of renal cells very low, and apoptosis normally is a rare event (Mene and Amore, 1998; Savill, 1994; Savill et al., 1996). However, the deficiency of SPRED2, a critical Ras/ERK/MAPK pathway inhibitor, may result in a disequilibrium between ERK-regulated cell proliferation, differentiation, and apoptosis. In this manner, FGFR3 mutations leading to a constitutive active RTK and an upregulated Ras/ERK/MAPK signaling cause thanatophoric dysplasia. This skeletal disorder is a severe and lethal form of dwarfism similar to the milder hypochondroplasia, which we observed in SPRED2 KO mice (Bundschu et al., 2005; Legeai-Mallet et al., 1998). Interestingly, FGFR3 mutations in thanatophoric dysplasia do not hamper chondrocyte proliferation but rather alter their differentiation by triggering premature apoptosis of chondrocytes (Legeai-Mallet et al., 1998). Similarly, missing inhibition of ERK pathways and premature apoptosis contribute to the dwarfism but could also play a role in the observed cell death in kidneys of SPRED2-deficient mice. Cell debris could evoke occlusions of the urinary system leading to the disturbed urine outflow and further resulting in hydronephrosis and kidney atrophy. Contrarily, hydronephrosis may also be the origin of apoptosis. The latter may occur in parallel to the hydronephrosis-provoked renal atrophy as described in several experimental rat studies using congenital and surgical hydronephrosis models (Gobe and Axelsen, 1987; Truong et al., 1996; Zhou et al., 2002). The demise of especially renal tubules rather than glomeruli, as also observed in SPRED2 KOs, seems to be a common consequence of hydronephrosis (Gobe and Axelsen, 1987; Truong et al., 1996; Zhou et al., 2002). While in case of premature apoptosis, due to upregulated MAPK signaling, the hydronephrosis would presumably rather develop bilaterally starting already in early embryonic development, the hydronephrosis observed in SPRED2 KO mice was mostly unilateral and developed in adulthood. Thus, in our SPRED2 mouse model renal apoptosis might be, together with the extreme parenchymal atrophy, a consequence of hydronephrosis. By visual inspection and dissection, we could not detect any obvious stones within or tumors outside the urinary system. Accordingly, the blood test results did not provide any indication for urolith development 2+ 3- because levels of Ca and PO4 , which may contribute to urinary stone formation, were normal (Table 1). Taken together, the exact mechanisms of hydronephrosis development and the resulting kidney deterioration have to be investigated further. However, the profound polydipsia of SPRED2 KOs might play a role because psyochogenic or congenital polydipsia have been shown to lead to hydronephrosis both in men and mice (Blum and Friedland, 1983; Blum et al., 1983; McDill et al., 2006; Silverstein et al., 1961).
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6.3. Polydipsia, Impaired Water Retention, and Hyperosmolality in SPRED2 KOs ‐ No Effect of SPRED2 on AVP‐ Dependent Water Balance Regulation
The polydipsia observed in our SPRED2 KO mouse model was characterized by nearly doubled daily water uptake. A main cause of polydipsia and resulting polyuria is an impaired ability of the kidneys to concentrate urine. Most frequent reasons for polydipsia are Diabetes mellitus, Diabetes insipidus, and other osmotic disorders leading to diuresis, e.g. defects in renal transport carriers required for reabsorption of ions or glucose. Diabetes mellitus is a well-known glucose metabolism disorder, which leads to osmotic diuresis due to increased glucose levels in serum and also in urine provoked by overload of renal glucose carriers required for glucose reabsorption. The elevation in serum glucose levels is caused by the lack of insulin or by an impaired cellular response to insulin, which normally regulates the glucose uptake from the blood into liver, skeletal muscle, and fat cells. In diabetes, blood glucose, usually the most readily available energy source in the body can not be used for metabolism. Instead, lipolysis takes place and fats are used as alternative energy source, which is accompanied by an increase in serum levels of cholesterol and triglycerides. However, because our SPRED2-deficient mice showed clearly reduced serum glucose, triglyceride, and cholesterol levels, we could exclude diabetes mellitus as cause of polydipsia. Diabetes insipidus exists in two different types, each with a different etiology. The most prevalent form, diabetes insipidus centralis, is caused by AVP deficiency, while the second form, diabetes insipidus renalis, is elicited by an insensitivity of the kidney epithelia to respond to AVP. We wondered if a lacking or insufficient production of AVP is causative for the polydipsia in the SPRED2 KO mice. AVP, by binding to V2Rs in the basolateral membrane, increases water permeability of the kidney's distal convoluted tubule and collecting duct by triggering the insertion of AQP2 water channels into the apical cell membranes. Water deprivation is a common experiment to investigate disorders in AVP production and release because under normal physiological conditions thirst leads to an increase in AVP secretion and water reabsorption (Chatelain et al., 2003; Sebaai et al., 2002). However, experimental water starvation for 48 h revealed no differences in AVP release of SPRED2 KO mice in comparison to WT controls. Thus, diabetes insipidus centralis does not seem to be causative for the augmented water consumption of SPRED2 KOs. Furthermore, SPRED-mediated Ras/ERK/MAPK signaling seems to play a negligible role in the regulation of AVP secretion. If SPRED2 is able to impact the development of diabetes insipidus renalis, which can amongst
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6. Discussion others be provoked by loss-of-function mutations of V2R (Yun et al., 2000) and AQP2 (Rojek et al., 2006; Yang et al., 2006), remains to be elucidated. Nevertheless, compared to the already profound body weight loss in WT controls, the body weights of SPRED2 KO mice were even further reduced after water starvation indicating an extreme loss of body fluid especially in KOs. Accordingly, due to body weight reduction, adrenal, kidney, and brain weight/body weight ratios were increased after water starvation, a phenomenon that is commonly observed in water deprivation experiments (Chatelain et al., 2003; Sebaai et al., 2002; Ulrich-Lai et al., 2006). While the absolute weights of kidneys and brains were kept almost constant during water deprivation, the adrenal weights even increased due to hyperplastic and hypertrophic events representing also a common consequence of water starvation and chronic stress (Chatelain et al., 2003; Sebaai et al., 2002; Ulrich-Lai et al., 2006). Enhanced fluid loss and impaired water retention in SPRED KOs were further reinforced because water deprivation also increased serum osmolality to a greater extent that in WT littermates. Additionally, serum osmolality in KOs was already augmented under basal conditions and even despite the elevated water uptake. This basal hyperosmolality that could not be compensated by enhanced drinking in SPRED2-deficient mice pointed towards a disorder affecting renal salt reabsorption, eliciting an elevated serum salt load and thus causing osmotic diuresis and polydipsia.
6.4. Hyperaldosteronsim – The Elicitor of Hyperosmolality and Salt and Water Imbalances in SPRED2 KO Mice
Indeed, enhanced drinking was accompanied by significantly elevated serum Na+ and Cl- levels, which were very likely causative for serum hyperosmolality in SPRED2 KO mice because the levels of all other determined osmolytes were unchanged (Table 1). Nearly congruent phenotypes to that of our SPRED2 KO mice were described in mouse models carrying loss-of- function mutations of the renal Na+-K+-2Cl- cotransporter (Takahashi et al., 2000), the ROMK channel (Lorenz et al., 2002), the V2R (Yun et al., 2000), and of AQP2 (Rojek et al., 2006; Yang et al., 2006). All these KO mouse models display polydipsia and/or polyuria, hydronephrosis, dramatically impaired urine concentrating abilities, and growth retardation. Furthermore, the loss-of-function models of the renal salt transporters (Lorenz et al., 2002; Takahashi et al., 2000) and the V2R (Yun et al., 2000) additionally showed elevated serum Na+ and Cl- levels and hyperosmolality as it was seen in combination with all the other symptoms also in the
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SPRED2 KOs. In these mouse models, hypernatremia, hyperchloremia, and the resulting hyperosmolality are regarded as secondary effects of dehydration due to an impaired salt reabsorption elicited by non-functional Na+-K+-2Cl- cotransporters (Takahashi et al., 2000) and ROMK channels, or due to an impaired water reabsorption elicited by non-functional V2Rs (Yun et al., 2000). However, in our SPRED2-deficient mice, elevated serum salt load does not seem to be a consequence of dysregulated salt and water transporters but instead caused by an imbalance in hormonal homeostasis. Hyperosmolality, elevated NaCl serum levels, and higher water uptake prompted us to estimate serum aldosterone and indeed, aldosterone levels were substantially increased in SPRED2 KOs. Hyperaldosteronism leads to Na+ and fluid retention in the kidney and thus to volume overload. Hypervolemia further results in the activation of counter-regulatory mechanisms, e.g. inhibition of the RAS and ANP release. This further causes increased renal excretion of salt and water and therefore leads to polyuria and polydipsia. Taken together, in our SPRED2 KO mice, aldosterone supports an increased Na+ reabsorption that can not be compensated by augmented water uptake. The resulting increased urinary sodium excretion additionally leads to osmotic diuresis and hence is the elicitor of the urinary concentrating defect, polyuria, and polydipsia. Thus, hyperaldosteronism seems to be causative for the disequilibrium of salt and water homeostasis in our SPRED2-deficient mice.
6.5. Increased Aldosterone‐Synthase Expression and Elevated Serum ACTH – Causes of Renin‐Angiotensin System‐ Independent Hyperaldosteronism
Hyperaldosteronism in our SPRED2 KO mice was characterized by nearly doubled serum aldosterone levels. Besides the disequilibrium of salt and water homeostasis described above, hyperaldosteronism might also be causally involved in the development of the observed kidney degeneration in SPRED2-deficient mice. Because hyperaldosteronism provokes the same symptoms as non-functional salt and water reabsorption mechanisms, e.g. Na+-K+-2Cl- cotransporter, ROMK, V2R, and AQP2, including hydronephrosis and kidney damage (Lorenz et al., 2002; Rojek et al., 2006; Takahashi et al., 2000; Yang et al., 2006; Yun et al., 2000), the elevated aldosterone levels in the SPRED2 KOs might contribute to hydronephrosis. In general, besides the well-known effects of aldosterone on the regulation of sodium and water homeostasis, aldosterone can also produce deleterious structural changes in tissues, especially heart and kidney. Aldosterone mediates vascular injury, hypertrophy, and dysregulation of 110
6. Discussion proliferation and apoptosis leading to fibrosis and tissue remodeling, all processes that might be involved in the kidney phenotype of SPRED2 KOs (Dooley et al., 2011; Hollenberg, 2004). The main stimulators of aldosterone release are K+, Ang II, and ACTH. An increase of serum K+, which can stimulate aldosterone release and thus aldosterone-mediated renal tubular K+ secretion, did not seem to be involved in the generation of hyperaldosteronism because K+ levels were rather decreased in SPRED2 KO animals. Dependent on the site of origin, hyperaldosteronism occurs in two forms: while the primary form is caused directly in the adrenal gland, where aldosterone biosynthesis takes place, the secondary form is provoked by overactivity of the RAS and hence is a result of excessive circulating Renin. Because the determination of Ang II levels revealed a decrease in serum Ang II by over 50% in the KOs, we conclude that hyperaldosteronism develops independently of an augmented RAS activity and thus is rather of primary nature. Aldosterone inhibits Renin release from the juxtaglomerular apparatus since it exerts negative feedback on the RAS indirectly by volume expansion (Kaufman et al., 1980). Renin secretion and hence the production of Ang II could be additionally impaired in SPRED2 KOs because Renin is produced by juxtaglomerular cells in the kidney (Tobian, 1962). Therefore, its synthesis and secretion might be disturbed in the atrophic kidney, which provides an additional explanation for the very low Ang II levels and RAS activity in the KOs. Primary hyperaldosteronism, also known as Conn´s syndrome, first has been described in 1955 and was caused by an aldosterone-secreting adenoma of the adrenal (Conn, 1955). Aside from increased serum aldosterone in parallel to a lower RAS activity, hypernatremia and hypokalemia are common consequences of primary hyperaldosteronism as also observed in our SPRED2 KO mice. However, the classic feature of hypertension is lacking. Blood pressure measurements by both the non-invasive tail cuff method in conscious mice and by invasive catheterization revealed that SPRED2 KO mice were normotensive until the age of 12 months (data not shown). Although aldosterone-induced volume overload leads to hypertension in most human patients, not all mouse strains develop hypertension in response to aldosterone. Especially C57BL/6 and C57BL/6 intercrosses like our SPRED2 KO mice, which are kept on a comparably mixed background, seem to be less susceptible to mineralocorticoid-induced hypertension (Hartner et al., 2003; Sontia et al., 2008). Furthermore, since normal mouse chow, which is mainly based on vegetable ingredients, contains low sodium but high potassium, it is likely that the common consequences of hyperaldosteronism, i.e. hypokalemia, hypernatremia, and hypertension are less pronounced in mice than in humans. Besides adrenal adenoma, further causes of primary hyperaldosteronism include adrenal carcinoma, glucocorticoid-remediable aldosteronism, and adrenal hyperplasia. In comparison to WTs, the adrenal weight/body weight ratios in SPRED2 KOs were significantly elevated and, although body weights were reduced due to dwarfism, the absolute weights of adrenals even 111
6. Discussion tended to be higher in the KO mice. This points towards a hypertrophic or hyperplastic event occurring in both adrenals. Indeed, in bilateral adrenal hyperplasia, which accounts for about 30% of all cases of hyperaldosteronism, the adrenal zona glomerulosa cells become hyperplastic resulting in excessive secretion of aldosterone (Davis et al., 1967). During aldosterone biosynthesis, the last steps from 11-deoxycorticosterone are mediated by aldosterone synthase, an enzyme exclusively expressed in the adrenal zona glomerulosa (Domalik et al., 1991; Ogishima et al., 1992). In our SPRED2 KO mice, immunohistochemical staining of aldosterone synthase in adrenal gland sections demonstrated an obviously augmented aldosterone synthase expression as compared to WT controls. A very similar phenotype is observed in female TASK1 (TWIK-related acid-sensitive K+ channel) KO mice, which resemble our SPRED2 KO mice in the increase and expansion of aldosterone synthase expression, hyperaldosteronism, hypokalemia, and diminished RAS activity (Heitzmann et al., 2008). In the TASK1-deficient mice, aldosterone synthase was absent the zona glomerulosa, the outer zone of the adrenal cortex, and the common site of aldosterone production. Instead, it was expressed to a greater extent than usually in deeper cortical zones corresponding to the zona faciculata and the zona reticularis. Similarly, in our SPRED2 KO mouse model, although aldosterone synthase expression was observed in the zona glomerulosa, the site of expression was obviously broadened. To our opinion, this increase in aldosterone synthase was either due to hyperplasia or hypertrophy of aldosterone synthase producing zona glomerulosa cells or due to repression of the zona fasciculata. The functional corticosterone synthesis and release from the zona fasciculata was attested by augmented serum corticosterone levels. In combination with the elevated adrenal weight/body weight ratios, in our opinion a hyperplasia or hypertrophy of aldosterone producing cells in the zona glomerulosa is more likely than to a mislocalization in the zona fasciculata. This assumption was further supported because SPRED2 expression in WT adrenals and endogenous Spred2 promoter activity in HET and KO adrenals were predominantly observed within the thin layer of the zona glomerulosa. Similar to TASK1, SPRED2 and the Ras/ERK/MAPK pathway might be involved in mechanisms required for the regular adrenocortical zonation and appropriate aldosterone synthase expression in the zona glomerulosa. In consequence of loss of SPRED2-mediated inhibition, the hyperplasia of aldosterone producing cells might be provoked by an upregulated Ras/ERK/MAPK signaling. In line with that, increased ERK phosphorylation has been shown to induce proliferation in adrenal zona glomerulosa cells in response to a multiplicity of stimuli, amongst others ACTH, Ang II, and FGF (Hoeflich and Bielohuby, 2009). In our mice, due to SPRED2 deficiency hyperproliferation of aldosterone-releasing cells might take place even independent of simulation, and overshooting ERK activity might be a possible cause of the observed bilateral adrenal hyperplasia and hyperaldosteronism in SPRED2 KOs.
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Besides augmented aldosterone expression, serum ACTH levels were found to be increased by 30% in the SPRED2 KOs. Aside from the trophic effect, which ACTH exerts on the adrenal gland alone or in combination with different stimuli and by activating amongst others the Ras/ERK/MAPK pathway (Hoeflich and Bielohuby, 2009; Ramachandran et al., 1977), it also mediates steroidogenic effects. Acutely, ACTH leads to the initiation of steroidogenesis and the mobilization of cholesterol, which is indispensable for the subsequent synthesis of corticosterone in the adrenal zona fasciculata and of aldosterone in the zona glomerulosa. Chronically, ACTH stimulates the transcription of all genes encoding the enzymes required for steroidogenesis (Sewer and Waterman, 2002, 2003), especially of 11β-hydroxylase, the critical enzyme in corticosterone synthesis (Kramer et al., 1983) but not of aldosterone synthase. In fact, aldosterone synthase expression is mainly stimulated by Ang II and K+, and especially chronic ACTH stimulation rather decreases aldosterone synthase transcription (Holland and Carr, 1993). However, in mice biosynthesis pathways of aldosterone and corticosterone are homologous up to corticosterone. Therefore, a combination of both the augmented ACTH levels and the elevated aldosterone synthase expression in adrenal zona glomerulosa cells might contribute to the hyperaldosteronism in our SPRED2-deficient mice. On the one hand, increased ACTH may lead to the mobilization of more cholesterol, which is required for the initiation of steroidogenesis and which results in a greater availability of steroidogenic aldosterone precursors, i.e. of pregnenolone, progesterone, 11-deoxycorticosterone, and corticosterone. On the other hand, the elevated number of aldosterone synthase-containing cells, which may be associated with increased ERK activity in the adrenals of SPRED2 KO mice, causes an enhanced aldosterone synthesis from the abundant precursors, i.e. 11- deoxycorticosterone, corticosterone, and 18-hydroxy-corticosterone.
6.6. Hyperactivity of the HPA Axis in SPRED2‐deficient Mice – Elevated Release of Stress and Growth Hormones
ACTH, due to its ability to stimulate especially 11β-hydroxylase, has a greater impact on glucocorticoid than on mineralocorticoid regulation (Kramer et al., 1983). Therefore, the hypercorticosteronism in SPRED2 KO mice, characterized by a more than doubled serum coricosterone level, is likely provoked by the elevated serum ACTH. Hypersecretion of ACTH is due to its role as stimulator of glucocorticoid synthesis associated with diseases affecting the glucocorticoid metabolism. One of them is Addison´s disease or primary adrenal insufficiency
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6. Discussion leading to hypocorticism due to adrenal dysgenesis, due to impaired functionality of steroidogenic enzymes, or due to autoimmune adrenal destruction. This insufficient glucocorticoid production leads to an impaired feedback regulation on ACTH release and hence to augmented serum ACTH. A second disease is Cushing’s disease, which describes the signs and symptoms of hypercorticism elicited by different causes. Cushing’s syndrome, the primary form of hypercorticism is caused by augmented cortisol production due to adrenal gland tumors, hyperplastic adrenal glands, or an excess of exogenously administered corticosteroids. However, the most common cause of Cushing's syndrome responsible for 70% of all cases is secondary hypercorticism. Here, augmented glucocorticoid levels are due to increased ACTH release, which is mostly caused by a benign pituitary adenoma or by excess production of hypothalamic CRH, the latter also called tertiary hypercorticism. Because elevated ACTH levels were accompanied by hypercorticosteronism, Addison´s disease seemed unlikely in our SPRED2-deficient mice, but we considered that they might display a mild form of Cushing´s disease. In a comparable mouse model, the deletion of the neuroendocrine protein 7B2, a binding protein of PC2, which is involved in the processing of ACTH, causes a lethal form of Cushing´s disease in mice (Westphal et al., 1999). Reminiscent of SPRED2 KO mice, circulating ACTH together with corticosterone was markedly elevated and associated with adrenocortical expansion and hypoglycemia (Westphal et al., 1999). Because hypothalamic CRH is the main endogenous stimulator of ACTH and thus indirectly also of the further downstream corticosterone, we determined CRH levels in brain lysates in order to enlighten ACTH overproduction. Since we detected a substantial elevation of hypothalamic CRH release by about 30% in SPRED2 KOs, we could demonstrate an upregulation of the entire HPA axis and of the released hormones CRH, ACTH, corticosterone, and aldosterone. In accordance to our SPRED2 KO mouse model, other transgenic mouse lines that overexpress CRH also exhibited augmented ACTH and corticosterone secretion associated with adrenal hypertrophy (Groenink et al., 2002; Stenzel-Poore et al., 1992) and displayed a Cushing´s disease-like phenotype (Stenzel-Poore et al., 1992). Vice versa, genomic ablation of CRH in mice resulted in low corticosterone levels, while levels of POMC and serum ACTH were unaltered. However, the diurnal variation of serum ACTH and corticosterone was completely absent, a phenomenon that might be involved in the observed atrophy of the adrenal cortex in this CRH KO mouse model (Venihaki and Majzoub, 1999). Thus, an appropriate regulation of HPA axis activity is required to maintain the morphology of the involved tissues, the circadian rhythmicity of stress hormone release, and the physiological responses to stress in general. Not only stress hormones are released from hypothalamus and pituitary in a sequential manner but also growth hormones. Similar to the hypothalamic CRH-stimulated pituitary ACTH release further resulting in adrenal corticosterone and aldosterone secretion, the hypothalamic release of growth-hormone-releasing hormone stimulates the secretion of GH from the anterior pituitary, 114
6. Discussion which further mediates the production of IGF-1 in the liver. Despite the hypochondroplasia-like dwarfism observed in the SPRED2 KOs, serum levels of both GH and IGF-1 were surprisingly found to be elevated. In our SPRED2 KO mouse model, the dwarf phenotype seemed to be caused by loss of SPRED2-mediated inhibition of FGFR3-dependent ERK activation (Bundschu et al., 2005). Accordingly, FGFR3 gain-of-function mutations are causative for different forms of dwarfism like achondroplasia, hypochondroplasia, and thanatophoric hyperplasia both in men (Bellus et al., 1995; Rousseau et al., 1994; Shiang et al., 1994; Tavormina et al., 1995) and mice (Chen et al., 1999a; Wang et al., 1999). Aside from FGFR3 signaling, a multiplicity of other mechanism regulate long bone growth, hereunder GH and IGF (Isaksson et al., 1982; Isgaard et al., 1988). Therefore, a common treatment of achondroplasia in humans is the administration of GH, which improves the disturbed bone growth because the GH-stimulated IGF-1 release prevents chondrogenic apoptosis induced by activating FGFR3 mutations (Koike et al., 2003). Taken together, on the one hand, overproduction of GH and IGF-1 might be a consequence of the generalized upregulation of the HPA axis in SPRED2 KO mice. On the other hand, it might represent an intrinsic mechanism to compensate the FGFR3-provoked defect in long bone growth. However, the increased GH and IGF-1 levels do not seem to be sufficient to overcome the hypochondroplasia-like dwarf phenotype of the SPRED2 KOs.
6.7. Excessive Grooming in SPRED2 KOs ‐ A Result of HPA Axis Overactivity and Evidence of Endogenous Stress
In addition to metabolic disorders, upregulation of stress hormone secretion from the HPA axis has also been shown to be involved in a variety of stress-associated and affective disorders, e.g. obsessive-compulsive disorder, depression, anxiety disorder, chronic pain, eat disorders, insomnia, and neurodegenerative disorders (Arborelius et al., 1999; Bao et al., 2008; Bomholt et al., 2004; Kluge et al., 2007; Stokes and Sikes, 1991). SPRED2 KO mice older than four months developed severe skin lesions, which were a consequence of excessive and injurious self-grooming. We assume that this enhanced grooming behavior results from an obsessive- compulsive disorder (OCD). OCD is a common psychiatric disorder and defined by the presence of persistent intrusive, obsessive thoughts and repetitive, compulsive actions often encompassed by anxiety and depressive symptoms (Miguel et al., 2005). Very strikingly, two mouse models carrying loss-of- function mutations of brain-specific proteins show a remarkable phenotypical similarity to our
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SPRED2 KOs. While the first mouse model lacks SAP90/PSD95-associated protein 3 (SAPAP3), a postsynaptic scaffolding protein highly expressed at striatal excitatory synapses (Welch et al., 2007), the second one is deficient of SLIT and NTRK-like protein-5 (SLITRK5), a neuron-specific transmembrane protein (Shmelkov et al., 2010). In agreement with our SPRED2 KO mice, these mouse lines develop bloody and purulent facial skin lesions and hair loss due to an obsessive-compulsive grooming disorder, which was associated with increased anxiety-like behaviors (Shmelkov et al., 2010; Welch et al., 2007). SAPAP3 and SLITRK5 are brain-specific, structural proteins, and thus it is not astonishing that their deficiency is associated with behavioral effects. However, SPRED2 is a Ras/MAPK/ERK signaling inhibitor but is highly expressed in brain and also involved in the regulation of CRH production and HPA axis activity. Indeed, CRH overexpressing mouse models are also susceptible to stress-associated diseases and anxiety-like behavior (Stenzel-Poore et al., 1994; van Gaalen et al., 2002). Similar to CRH overexpression, intracerebroventricular administration of CRH produces a significant increase in grooming, rearing, and locomotor activity in rat, which is consistent with its role as a mediator of the stress response (Dunn and Berridge, 1990; Sutton et al., 1982). Hence, the observed obsessive-compulsive grooming of our SPRFED2 KO mice is very likely a consequence of the excessive endogenous stress caused by overproduction of hormones released from the HPA axis.
6.8. SPRED2 ‐ A Novel Regulator of CRH Gene Transcription and Release
SPRED1 has been shown to be expressed predominantly in neural tissues, i.e. in brain and in the central nervous system (Engelhardt et al., 2004; Kato et al., 2003). There, it participates in cortical development, neural stem cell proliferation, and vesicular trafficking (Engelhardt et al., 2004; Phoenix and Temple, 2010). In addition to a generally similar expression pattern of SPRED2 in brain (Bundschu et al., 2006a; Engelhardt et al., 2004), we could demonstrate both Spred2 promoter activity and SPRED2 expression especially in hypothalamus and pituitary gland. Both tissues are part of the HPA axis and the release of HPA stress hormones from these tissues is triggered by the limbic system. The latter contains a multiplicity of brain structures including the limbic cortex, hippocampus, amygdala, and also the hypothalamus, which initiates HPA axis activity by CRH release.
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6. Discussion
Based on SPRED2 expression in the hypothalamus, which is in parallel the most upstream trigger of the HPA axis, based on the elevated hypothamalmic CRH release in the SPRED2 KOs, and based on the described functions of SPREDs as Ras/MAPK/ERK pathway inhibitors, we aimed to unravel the mechanism how SPREDs could be involved in the regulation of CRH release and HPA axis activity. Investigation of MAPK/ERK activity in PVN region-containing brain lysates of SPRED2 KO mice revealed a significantly increased ERK phosphorylation. This clearly demonstrated an upregulated hypothalamic Ras/ERK/MAPK signaling as a consequence of the lacking suppressive effect of SPRED2. Because the Ras/ERK/MAPK cascade is well-known to be involved in transcriptional regulation of a great variety of target genes, we considered that it might also have an impact on CRH transcription. With our studies using a CRH promoter reporter containing the RE-1/NRSE regulatory element, we could confirm that CRH transcription in general is dependent on the RE- 1/NRSE, which represses transcriptional activity of the CRH promoter (Seth and Majzoub, 2001). In studies using a CRH promoter reporter lacking the RE-1/NRSE, we could show that under these conditions the CRH promoter is active and that SPREDs indeed are able to inhibit CRH promoter activity and transcription in hypothalamic mHypoE-44 cells. In vivo, in hypothalami of SPRED2-deficient mice, levels of CRH mRNA were accordingly higher. Amongst others, activation of CRH transcription involves the phosphorylation and nuclear translocation of CREB and its recruitment to the CRE (cAMP responsive element) on the CRH promoter (Seasholtz et al., 1988). Multiple kinases can phosphorylate CREB, such as PKC, Ca2+/calmodulin-dependent protein kinase (CaMK), PKA, but also MAPKs. Indeed, in hypothalamic PVN neurons producing CRH, the Ras/ERK/MAPK signaling pathway is activated in response to various stimuli, e.g. chronic stress, catecholamines, hypoglycemia, lipopolysaccharides, and prolactin. This increase in ERK phosphorylation induces an activation and phosphorylation of downstream transcription factors in the hypothalamus (Blume et al., 2009; Khan and Watts, 2004; Singru et al., 2008) and in the limbic system (Gerrits et al., 2006). It also provokes a parallel activation of CRH transcription in these neurons (Blume et al., 2009; Khan and Watts, 2004; Singru et al., 2008). Thus, we have confirmed that the Ras/ERK/MAPK cascade is in fact a potent regulator of CRH promoter activity both in vitro and in vivo and hence identified SPREDs as novel suppressors of CRH transcription. In order to enlighten, which of the various target transcription factors might be the mediator between Ras/ERK/MAPK pathway activation and induction of CRH transcription, we performed a second reporter assay in mHypoE-44 cells, which was able to demonstrate the effect of SPREDs on Ets-dependent transcription. The Ets transcription factor family was of special interest for us because the CRH promoter comprises at least four binding sites for Ets,1 and because Ets1 is a well-known target of Ras/ERK/MAPK signaling (Wasylyk et al., 1998). In fact, in our reporter assay, SPREDs were also able to suppress Ets1-mediated transcription. The Ets 117
6. Discussion protein family is one of the largest families of transcription factors and contains a highly conserved DNA-binding domain, the Ets domain. It is common for all family members, which are sub-classified into twelve subfamilies comprising together at least 27 members in mammals (Sharrocks, 2001). The structure of the Ets domain is a variant of the winged helix-turn-helix motif and binds specifically to 11 bp DNA sequences that comprise a central GGA motif. That especially MAPK pathways have been linked with numerous regulatory events that involve Ets domain transcription factors is underlined by the identified docking domains for MAPKs in Ets domain proteins. For ERK, these special docking sites have been identified in the Ets family transcription factors Lin1, Elk1, and Ets1 (Jacobs et al., 1998; Jacobs et al., 1999; Tan et al., 1998; Yang et al., 1998a; Yang et al., 1998b). Taken together, Ets transcription factors, due to their multiple family members and even greater number of targets, regulate various developmental processes, e.g. neurogenesis, hematopoiesis, and angiogenesis (Sharrocks, 2001). Their involvement in HPA axis activity and CRH transcription, however, has been identified in this study first. Aside from CRH promoter activity and transcription, also CRH production and release was influenced by SPREDs. While CRH secretion was inhibited by SPREDs in mHypoE-44 cells in vitro, it was increased in vivo in SPRED2-deficient mice. In sum, we could demonstrate that SPRED2 is not only expressed in tissues of the HPA axis but also plays a critical role in the regulation of the HPA axis. Thus, the loss of SPRED- mediated Ras/ERK/MAPK suppression in our SPRED2 KO mice elicits an upregulated hypothalamic ERK signaling. This is followed by an augmented Ets-dependent CRH transcription and release and results in an overproduction of stress hormones at all levels of the HPA axis. The dysregulation of HPA hormone secretion has very far reaching consequences for the complete body. Therefore, the identification of SPRED2 as a critical regulator of HPA axis activity is a very valuable explanation, why SPRED2 deficiency leads to the observed very complex and versatile phenotype in mice, affecting multiple organs, hormonal, water, and electrolyte homeostasis, and mood.
6.9. SPRED1 and SPRED2 Deficiency ‐ Redundancy in Protein Function and Impact on Cancer and Rasopathies
While SPRED3 expression has been described exclusively in brain, most tissues show overlapping expression of both SPRED1 and SPRED2. SPRED1, which is found predominantly
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6. Discussion in neural tissues, is also detectable in most other organs similar to SPRED2, which is expressed almost ubiquitously throughout the whole body (Bundschu et al., 2006a; Engelhardt et al., 2004; Kato et al., 2003). While in mice, neither the sole deficiency of SPRED1 nor that of SPRED2 affects embryonic survival, SPRED1/2 double-deficient mice suffer from embryonic lethality due to severe subcutaneous hemorrhage and edema (Taniguchi et al., 2007). Thus, overlapping expression and overlapping protein functions might explain the viability and the comparatively mild phenotype of single SPRED1 (Denayer et al., 2008; Inoue et al., 2005; Nonami et al., 2004; Phoenix and Temple, 2010) and single SPRED2 KO mice (Nobuhisa et al., 2004; Wakabayashi et al., 2012). A comprehensive phenotypical characterization is not available for the SPRED- deficient mouse models generated by the other groups, and no serious impairments of these mouse models have been published. Nevertheless, the phenotype of the SPRED2 KO mouse model generated in our lab (Bundschu et al., 2005) seems to be particularly versatile and complex, especially in mice older than three months (Ullrich et al., 2011). In comparison to the SPRED2 KOs from the Japanese group, the putative more severe phenotype of our SPRED2 KO mice might be on the one hand dependent on the genetic background. While the Japanese SPRED1 and SPRED2 KO mice were backcrossed to C57BL/6 and are kept on a defined genetic background, the SPRED2 KO mouse line generated by us is kept on a mixed C57BL/6 x 129P2/OlaHsd background. On the other hand, the expression of the EVH1-β-geo fusion protein especially in our mouse model might play a role. The function of this fusion protein, i.e. if it might exert any dominant negative effect, is still a point of discussion and it might be dependent on the tissue and the regulated process (6.1.). Due to the embryonic lethality of conventional SPRED1/2 double-deficient mice, it has been impossible to study the function and contribution of individual SPRED family members to the integrity of special organs. In order to investigate both the specific and the common functions of the different SPREDs, the generation of conditional KO mice is indispensable. Conditional SPRED KO models would also allow to discriminate between effects caused by a single Spred1 or Spred2 gene deficiency in a specific organ and to exclude systematic effects, comorbidities, and affection of other organs present in our global SPRED2 KO mouse model. Therefore, we already cloned SPRED1 and SPRED2 KO targeting vectors based on the Cre-loxP-system and allowing the generation of both global and conditional KOs. By mating our floxed SPRED mouse lines with tissue-specific Cre-expressing mouse lines, we will be able to investigate specific SPRED functions in a tissue-specific background. Additionally, due to the great potential of the targeting vectors to vary the SPRED1 and SPRED2 KO strategies, and due to the ability to mate the different conditional and/or global SPRED1 and SPRED2 KO mouse models with each other, we will be able to investigate all possible genotype combinations. The consequence, which its deficiency causes in humans, is a very important question especially regarding SPRED2 protein function. SPRED1 deficiency in humans has been linked 119
6. Discussion to neurofibromatosis type 1-like Legius syndrome characterized predominantly by café-au-lait spots on the skin, facial abnormalities, and behavioral and learning problems (Brems et al., 2007; Messiaen et al., 2009; Pasmant et al., 2009b; Spurlock et al., 2009). In fact, SPRED1 KO mice very strikingly reflect the phenotype of Legius syndrome patients because they also show learning disabilities (Denayer et al., 2008) and facial dysmorphism (Brems et al., 2007; Inoue et al., 2005). Thus, our SPRED2 KO mouse model likely demonstrates also classical features of germ-line loss-of-function mutations of SPRED2 in humans. Interestingly the reduced body weight and the facial abnormalities described for the SPRED1 KOs (Brems et al., 2007; Inoue et al., 2005) may relate to dwarfism and the shortened face seen in our SPRED2 KO mice (Bundschu et al., 2005). Consistent with the elevated ACTH levels in SPRED2 KOs, SPRED1 deficiency leads to the development of café-au-lait spots in the skin. These brown macules contain melanin deposits probably caused by elevated levels of α-MSH, a cleavage product of ACTH and a stimulator of melanin production and release by melanocytes in skin and hair (Schwindinger and Levine, 1993). Besides the lethal phenotype of SPRED1/2 double KO mice (Taniguchi et al., 2007), these are further hints towards functional redundancy for SPRED1 and SPRED2. Because Legius syndrome belongs to the rasopathies, a group of diseases all caused by a hyperactivated Ras/ERK/MAPK signaling, SPRED2 deficiency very likely also results in a rasopathy-like disease pattern. Characteristic symptoms very strongly reminiscent of the rasopathies (Tidyman and Rauen, 2009) and also present in our SPRED2 KO mice are short stature, recognizable facial features, renal anomalies, and cardiac disorders (hypertrophy, arrhythmias; data not shown). There are further indications for developmental delay, mental retardation and learning issues, and malabsorption; however, these assumptions have to be verified by suitable experiments. Furthermore, the consequences of SPRED2 germline loss-of- function mutations are still unknown but a genetic screen for SPRED2 mutations in DNA samples from rasopathy patients has been initiated by us and is in progress. A large number of Ras/Raf/ERK pathway components were initially identified as proto- oncogenes, e.g. overexpression or activating mutants of growth factors like FGF (Delli Bovi et al., 1987), growth factor receptors, e.g. EGFR (Velu et al., 1987), Ras (Chang et al., 1982a; Chang et al., 1982b), Raf (Rapp et al., 1983), and various transcription factors targeted by the Ras/ERK/MAPK pathway, amongst others the above mentioned c-Fos (Miller et al., 1984), c- Jun (Maki et al., 1987), NF-κB (Kieran et al., 1990), c-Myc (Roussel et al., 1979; Weiss, 1982), and Ets1 (Watson et al., 1985). In some cases, rasopathy patients show an elevated predisposition to benign tumors like neurofibromas, Lisch nodules, lipomas but also to malignancies, e.g. astromas, gliomas, leukemias (Brems et al., 2007; Messiaen et al., 2009; Pasmant et al., 2009a). However, although the deficiency of SPREDs also leads to a
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6. Discussion consecutive upregulation of Ras/Raf/ERK signaling, it basically does not result in the development of tumors or a special form of cancer.
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Conclusion and Outlook 7
With this study, we initially demonstrated that SPRED2 is indispensable for appropriate regulation of HPA axis activity and body homeostasis. For the first time, we could show that SPRED2, by downregulating MAPK signaling via inhibition of ERK phosphorylation, suppresses Ets transcription factor-dependent activation of the CRH promoter and thus reduces CRH transcription and release (Figure 35). In line with that, our SPRED2 KO mice show an upregulated ERK activity in PVN-containing hypothalamic brain regions accompanied by both increased hypothalamic CRH mRNA levels and elevated release. The inhibitory effect of SPREDs could be confirmed by in vitro studies using the hypothalamic cell line mHypoE-44 and showing that both SPRED1 and SPRED2 are able to suppress Ets factor-dependent transcription, CRH promoter activity, and CRH secretion. This elevated production of CRH, the most upstream regulator of the HPA axis, elicits an elevated hormone secretion from all levels of the HPA axis, including downstream pituitary ACTH and adrenal corticosterone and aldosterone. This HPA axis upregulation resulting in hypercorticism is very likely causative for the obsessive grooming behavior and the consecutive wound development in SPRED2 KO mice. HPA axis hyperactivity in combination with the augmented adrenal aldosterone synthase expression induces hyperaldosteronism leading to salt and water imbalances (Figure 35). Due to the increase of serum aldosterone, the observed hypernatremia, hyperchloremia, and hyperosmolality can not be compensated by augmented water consumption. Water deprivation showed a substantial higher loss of body fluid in SPRED2 KOs and that polydispia develops independent of an AVP release disorder. Excessive drinking might causally be involved in the development of hydronephrosis further associated with renal atrophy, lymphocytic infiltration, apoptosis, and kidney damage. The complex and manifold phenotype of our SPRED2 KO mice might be further reinforced by the putative dominant negative effect of the expressed EVH1-β- geo fusion protein.
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Figure 35: Putative Mechanism How SPRED2 Controls Hypothalamic-Pituitary-Adrenal Hormone Secretion In our SPRED2 KO mouse model, due to the loss of SPRED2-mediated inhibition, Ras/ERK/MAPK signaling is upregulated in parvocellular neurons of the hypothalamic PVN. Increased ERK phosphorylation and activity leads to increased activation of Ets1 transcription factors, which in turn bind to Ets1 binding sites in the CRH promoter region and enhance CRH transcription. The resulting augmented CRH-release from the hypothalamus induces a complete upregulation of hormone secretion from the HPA axis, including elevated pituitary ACTH, adrenal corticosterone, and adrenal aldosterone release. The hyperaldosteronism develops independently of the RAS, as seen by downregulated Ang II levels, but is a consequence of HPA axis overactivation and of increased aldosterone synthase expression in adrenals of SPRED2 KO mice. This dysregulation of hormonal cascades causes both salt and water imbalances associated with kidney deterioration and altered stress-associated behavior in SPRED2 KO Mice. Thus, our study identifies SPREDs as novel regulators of hormone secretion and body homeostasis. Figure in part reproduced from http://histoblog.viabloga.com/images/hypophyse_1_t.800.jpg, form http://www.gru.edu/mcg/phy/raineylab/images/ webfigure2.jpg, and from http://www.paradisi.de/images_artikel/1/12556_0.jpg.
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7. Conclusion and Outlook
Taken together, our study indicates that missing SPRED2-dependent inhibition of the Ras/ERK/MAPK pathway contributes to overshooting HPA hormone production, to the pathogenesis of hyperaldosteronism, and to obsessive-compulsive disorders.
7.1. SPRED2 – A Critical Mediator of Synaptic Transmission and Regulator of Behavior?
Based on the overactivated HPA axis and the obsessive grooming of our SPRED2 KO mice, we performed further studies to investigate how the elevated endogenous stress response impacts behavior, emotional state, and brain structure and functions. Because excessive stress hormone release from the HPA axis is associated with a great variety of behavioral and mood disorders in humans and mice (Arborelius et al., 1999; Bao et al., 2008; Bomholt et al., 2004; Kluge et al., 2007; Stokes and Sikes, 1991), we performed a set of behavioral standard tests with our SPRED2 KO mice and WT littermate controls, including elevated plus maze, open field, light dark box, and forced swim test. All these tests especially investigating anxiety and depression indicated a reduced anxiety in the SPRED2 KOs, which correlated with increasing age and the incidence of compulsive grooming. The stress hormone production of the HPA axis is subject to the control of neural afferents, which project from structures of the limbic system such as amygdala to the hypothalamus. The amygdala projects amongst others to the hypothalamic PVN from where CRH release takes place. It is instrumental in the development of anxiety and plays an important role in emotional evaluation and recognition of situations. Because incoming neural inputs to the amygdala are often of thalamic origin and because SPRED2 expression could be confirmed in both thalamus and amygdala, we performed electrophysiological measurements on acute brain slices to study synaptic transmission from thalamic nerve fibers to the amygdala. In our SPRED2 KO mice, we detected elevated excitatory postsynaptic currents (EPSCs), and the stimulation current required for triggering an action potential was reduced. Further studies revealed an increased synaptic depression and decreased frequencies and amplitudes of spontaneous vesicle fusions. In order to unravel if these changes in thalamic-amygdalal synaptic transmission are either due to pre- or postsynaptic events, we performed expression studies of both characteristic pre- and postsynaptic proteins by quantitative RT-PCR and Western Blot. Both amygdalal mRNA and protein levels of the postsynaptic metabotropic glutamate receptors mGluR2 and mGluR5 and the postsynaptic density protein 95 (PSD95) were accordingly upregulated, whilst that of presynaptic bassoon was downregulated. These data suggest counter-regulating mechanisms 125
7. Conclusion and Outlook between presynaptic and postsynaptic activities reflected by the dysregulated protein expressions. Another indication for a functional role of SPREDs in synaptic transmission is the confirmed colocalization of SPREDs with vesicles (Engelhardt et al., 2004; Phoenix and Temple, 2010) and their interaction with tubulin because microtubules amongst others mediate vesicle transport. Recently, the interaction with both α- and β-tubulines has been detected by us in a GST-EVH1-SPRED2 pulldown assay and confirmed by a tubulin-dimer pulldown assay. Electron microscopy studies demonstrated the general structural integrity of amygdalal synapses; however, detailed investigations are needed to determine e.g. the number and size of vesicles, dendritic counts, and the postsynaptic density. Because fluoxetine is a common drug for the treatment of mood disorders, especially obsessive-compulsive disorders and depression, we tried to rescue the excessive grooming by oral fluoxetine treatment. The antidepressant of the selective serotonin reuptake inhibitor class was administered at a dose of 20mg/kg/day within white chocolate and for the duration of four weeks. In fact, photo documentations on day zero, after two weeks, and after four weeks demonstrated a reduced severity and occurrence of wounds. In line, video documentations in the same time intervals also indicated a reduced grooming behavior after fluoxetine treatment, which further underlines the existence of a mood disorder in our SPRED2 KOs. Taken together, this continuation study demonstrated that SPRED deficiency results in obsessive-compulsive grooming and in altered anxiety behavior, which is associated with increased synaptic transmission from thalamus to amygdala and can be rescued by fluoxetine treatment. Both efferent amygdalal nerve fibers, which trigger hypothalamic CRH release (Herman and Cullinan, 1997), and CRH, which binds to CRH1 receptors also abundant in the amygdala (Potter et al., 1994), may contribute to the behavioral disorders in the SPRED2 KOs. Increased synaptic transmission is accompanied by altered expression of pre- and postsynaptic proteins in the amygdala, very likely again mediated by loss of SPRED-dependent transcriptional inhibition of the dysregulated proteins. Thus, our study indicates a causal context between SPREDs, Ras/ERK/MAPK signaling, and obsessive-compulsive disorders for the first time and describes novel and detailed function of SPREDs in the brain.
7.2. SPRED2 – A Novel Regulator of Cardiac Hypertrophy and Electrical Conduction in the Heart?
A second study was conducted on the basis of the observation that dissections of SPRED2 KO mice older than three months did not only demonstrate kidney damage but also obviously 126
7. Conclusion and Outlook enlarged hearts. Calculated heart weight/body weight ratios were also elevated in the SPRED2 KOs. This was not a secondary effect of dwarfism because average absolute KO heart weights in comparison to that of WTs, which have an increased average body weight, were even slightly augmented. Thus, this was clear indication for cardiac hypertrophy. Because the manifest hyperaldosteronism and the reduced survival probability also indicate a cardiac phenotype, we investigated the cardiac performance of SPRED2 KO mice in a comprehensive and systematic manner by invasive and non-invasive standard procedures. Echocardiographic measurements performed in papillar and apical views and in systolic and diastolic dimensions confirmed the increased heart weight/body weight ratio by revealing an increased thickness of left-ventricular posterior walls in SPRED2 KO mice. Accordingly, the end-systolic and end-diastolic inner diameters of the left ventricle were decreased. These echocardiographic investigations, which allow the precise measurement of cardiac dimensions, were further confirmed and supplemented by invasive hemodynamics allowing real time measurements of left-ventricular pressure volume loops. Hemodynamics additionally and unexpectedly revealed an increased stroke volume, an elevated ejection fraction, and an augmented peak rate of pressure rise. Associated with this, calculated cardiac output and cardiac index were also increased in SPRED2 KOs. In order to be able to determine the heart rate, to investigate electrical conduction, and to detect possible arrhythmias, we performed high resolution electrocardiographs (ECGs) both under resting and stress conditions. These ECGs demonstrated an accelerated heart rate already under basal conditions, which together with the elevated stroke volume contributes to the increased cardiac output. Under stress simulation by isoproterenol administration, the heart rate between KOs and WTs did not differ. The increase of basal heart rate was characterized by a shortened TP segment in the SPRED2 KO mice, which reflects the time between ventricular repolarization and atrial depolarization. This is likely caused by premature depolarization of pacemaker cells, while the resulting positive chronotropic and inotropic effect did not seem to be beneficial for the SPRED2 KO mice. In sum, ECG recordings demonstrated that SPRED2 KOs are prone to develop various forms of spontaneous arrhythmias, e.g. different forms of AV blocks and extrasystoles. To investigate the susceptibility of KOs to develop arrhythmias also under stress conditions, we applied intracardiac overdrive pacing using an octapolar electrophysiological catheter. Pacing of atriae at decreasing pulse pause frequencies and increasing heart rates (50, 33, 25, 20 ms ≙ 1200, 1800, 2400, 3000 bpm) elicited atrial fibrillation in KOs starting at a pacing rate of 1800 bpm, whereas electrical conduction was not affected in WT controls under the same stimulus- provoked conditions. Furthermore, atrial pacing at a heart rate of 700 bpm indicated impaired sinus node function by prolonged sinus node recovery times in the SPRED2 KOs. Profound SPRED2 expression in the heart has been demonstrated on mRNA level by RT-PCR and on protein level by Western blot. X-Gal-stainings further detected Spred2 promoter activity in both 127
7. Conclusion and Outlook atriae and ventricles. Upregulation of Ras/ERK/MAPK signaling in the heart is a critical marker of cardiac hypertrophy. In fact, the deletion of the ERK/MAPK inhibitor SPRED2 leads to cardiac hypertrophy and an increased cardiac ERK phosphorylation, which was determined by Western blot of heart lysates. In order to examine if cardiac dysfunction is caused directly by ablation of SPRED2 or indirectly by SPRED2 deficiency-mediated hyperaldosteronism, we rescued the hyperaldosteronism by treatment of SPRED2 KO mice and WT littermate controls with the aldosterone antagonist eplerenone. Eplerenone was administered by normal chow at a dose of 100mg/kg/day. All the above mentioned experiments to characterize cardiac performance were repeated in the same manner with the eplerenone-treated mice. Although the data analyses are still in progress, the heart weight/body weight ratio of the rescued mice is still elevated in SPRED2 KOs and does not significantly differ from the untreated groups, which indicates that the cardiac phenotype is at least to a great extent mediated by SPRED2 deficiency. In sum, SPRED2 deficiency in mice provokes cardiac hypertrophy, which is confirmed by the increased heart weight/body weight ratio, the elevated left-ventricular wall thickness, the accordingly decreased systolic and endsystolic diameters, and the overactivated Ras/ERK- MAPK cascade. Surprisingly, cardiac ouput and index were higher in the SPRED2 KOs, which is caused by elevated stroke volume/ejection fraction and heart rate, the latter presumably caused by facilitated or premature excitation of pacemaker cells. The resulting positive chronotropic and inotropic effect is not beneficial for the SPRED2 KO mice since it is accompanied by arrhythmias both under resting and stress conditions. The causative mechanisms are still under investigation and hyperaldosteronism alone does not seem to be responsible for the cardiac phenotype of our SPRED2 KOs. Thus, this study might identify SPREDs as novel modulators of cardiac hypertrophy and electrical conduction in the heart.
7.3. SPRED2 Deficiency – A Cause of Rasopathy in Humans?
Apart from our SPRED2 KO model, which is undisputedly a very useful tool to study Spred2 gene function in vivo, we are engaged in correlating our mouse data with a human disease pattern. Human loss-of-function mutations of SPRED1 cause Legius syndrome, which is very similar to neurofibromatosis and belongs to the disease group of rasopaties. Therefore, we perform exon screening of SPRED2 mutations in DNA samples from rasopathy patients in cooperation with the Department of Human Genetics at the Universitaetsklinikum Hamburg- Eppendorf. Patients showing characteristic features of rasopathy syndromes, e.g. that of Costello syndrome, but lacking a characteristic mutation in the described disease causing gene,
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7. Conclusion and Outlook i.e. in H-Ras, are of special interest for us. Based on the phenotype of our SPRED2 KO mouse model, which is already indicative for a rasopathy-like disease, and based on our recently initiated genetic screen for germline loss-of-function in humans, we are confident to identify a human correlate of SPRED2 deficiency in the near future.
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Appendix 9
9. 1. Genotyping
Murine Spred2 Gene with and without the pGT0 Gene Trap Vector behind Exon 4 and Appropriate Primers Used for Amplification of Specific WT and KO Fragments
Sequence Origin: ENSEMBL Database Chromosome 11: 19,924,442 - 20,022,577, Genomic DNA
WT DNA Fragment (803 bp): 164 bp – 966 bp (Pr.1 – Pr.2)
Legend: Spred2 - Intron 4 Primer SPR2-Geno-WT/KO-Forward, Pr. 1 Primer SPR2-Geno-WT-Reverse, Pr. 2:
GAGACAGGGTTTTTCTGTGCGGCCTTGGCTGTCCTGGAACTCAATCAGAAGGACCATGCTAGCC TTGAACTCACTGAGATCTGCTGGGACTAAATGCATGCACCAGCAGCACCCAGCTAACAAATAGT TTTCTCTCAGTCATCAGGCTGCCTTCTCGGCCCCAGCTTGACCGGCACCCCGGTGAGCACAGGC TCACCTCAGTAGTGTTGATCTCTTGTTAGTCAGTTGACTCTTGAGCCCTGATGATTAGCATACA CACATTTTTAGCCAAAGTATTACAATAACATCCCTTACTCCAAGTCTAGAGAGTGTCTTTGGTA CCCTCTGAAACCCAGAGGACCAGGCCTCCATTGTCCATGTTTCTCTCAGAATTCTTAGCTTCCA AGATCTCTCGAGAATGACCCATTAAGTTTTCAGTCCTCACCAGGAAAAAGCCAATCCCTGAGAG 157
9. Appendix
TAAAGGACAGCCAGGGCTGCAGAGACAAACCCTGCTGTCTCAGAAGGGAGTGGGGACGGGCTTT TAGTTCTACTTGTCATAGTGTCTGGAAGCCAAGGTCAGGAATGGATCAACTATGCCCAGTCACA GGAGAGTGGCTGGTAGGAGCCAAGTATCTATCTTCCACCATGGCCCAGTACTGTTTTTACATTT AGAAAACCAGCTTGTCTTTCCAAGGCCTTGCTGCTGGGCCTCTCCAACCATTGAGGTAACTACT TCATTCACATTGAGACATTCTGTTCATTCTCATGTCTAAGACAACTCAAGACTGGAAAGCCAGG AGTGATGGTTCATATTCTATAATCCCAGCACTTGAAATGTAAAGTCTAAAAACCAAGCCAGTGT GTGGGGGTGTGGTGGGGGCAGAGACAGAGAGATGAGATATGGCTCAAAGGTTAAAAGCACCTAC TACTACTACTAGCACCCAGCTAAGATGGCTCACAACCACCCACCTATAAGCTCCAAGGGATCCA GTGCCCTCTGCTGCCTCCATGGGCACCTACACTCAAATGCACATTATTAAAAATGAAATAAACT AGGCCCATGGAGGCCAGAAAAGAGCATCAGCTCCCCCCAGACCTGGAGTTATAGGCAGTCGAAG CTGTCTGTAGGTGGTGAGAATCTGGGCATTCAAGTCTGCTCACCCCGGGGCCCCTTCCCGCCCA TACCTTTAAT
KO DNA Fragment (452 bp): 164 bp – 615 bp (Pr.1 – Pr.3)
Legend: Spred2 - Intron 4 XXX: Unknown Sequence pGTO pfs Gene Trap Vector -geo CDS Primer SPR2-Geno-WT/KO-Forward, Pr. 1 Primer SPR2-Geno-KO-Reverse, Pr. 3: Primer SPR2-Geno-WT-Reverse, Pr. 2:
GAGACAGGGTTTTTCTGTGCGGCCTTGGCTGTCCTGGAACTCAATCAGAAGGACCATGCTAGCC TTGAACTCACTGAGATCTGCTGGGACTAAATGCATGCACCAGCAGCACCCAGCTAACAAATAGT TTTCTCTCAGTCATCAGGCTGCCTTCTCGGCCCCAGCTTGACCGGCACCCCGGTGAGCACAGGC TCACCTCAGTAGTGTTGATCTCTTGTTAGTCAGTTGACTCTTGAGCCCTGATGATTAGCATACA CACATTTTTAGCCAAAGTATTACAATAACATCCCTTACTCCAAGTCTAGAGAGTGTCTTTGGTA CCCTCTGAAACCCAGAGGACCAGGCCTCCATTGTCCATGTTTCTCTCAGAATTCTTAGCTTCCA AGATCTCTCGAGAATGACCCATTAAGTTTTCAGTCCTCACCAGGAAAAAGCCAATCCCTGAGAG 158
9. Appendix
TAAAGGACAGCCAGGGCTGCAGAGACAAACCCTGCTGTCTCAGTGTTATCACTCATGGTTATGG CAGCACTGCATAATTCTCTTACTGTCATGCCATCTGTAAGATGCTTTTCTGTGACTGGTGAGTA Ctctttcccagggagggaaggaggagagtttgagatccgctccggaagtcggggttcaggtttg agcaggccaggcctctcccgtggtctcgccctcttgtcctagaagcctcactggccaggtgtaa gccaggtcgtgggtgccgagccctgctccctcatcctcagcatggatgtgaagaggactgtatg gcgtgcgggtgtgtgtgaccgtgggtacacttaaaacaccgggttttggatctgcactgtcccg gatgtcctctggtgctcaaagacccttttgggtttgccctttggtaagagcgccgggatctact tgtctggaggccagggagtcctcagccgaggcttgccgcccctgactgcactgcactgagtagt ggatgggagagtctggtaccgcactgccggtttcctccaccatccccgcagcgcagggcagtgc attccgtcctggctgcgaagggggatggtcgggccttctccagcctcttccgcttctagcgaag gggccttgatggaagggcccgcatgtctccaaagttgattcatgcttcttgcacagagaaagac cagaaagaaggtctcaagttttagccggtagcccggatggccttttcctgcacggcaccatatg aaccttgtgaccctgactttgagacccctctaacccaaggcccctaccactttaccctttccct ttgaaggctttcccacaccaccctccacacttnccccaaacactgccaactatgtaggaggaag gggttgggactaacagaagaacccgttgtggggaagctgttgggagggtcactttatgttcttg cccaaggtcagttgggtggcctgcttctgatgaggtggtcccaaggtctggggtagaaggtgag agggacaggccaccaaggtcagccccccccccctatcccataggagccaggtccctctcctgga caggaagactgaaggggagatgccagagactcagtgaagcctggggtaccctattggagtcctt caaggaaacaaacttggcctcaccaggcctcagccttggctcctcctgggaactctactgccct tgggatcccttgtagttgtgggttacataggaaggggacggattccccttgactggctagccta ctcttttcttcagtcttctccatctcctctcaccgttctctcgaccctttccctaggatagact tggaaaaagataaggggagaaaaacaaatgcaaacgaggccagaaagattttggctgggcattc cttccgctagcttttattgggatcccctagtttgtgataggccttttagctacatctgccaatc catctcattttcacacacacacacaccactttccttctggtcagtgggcacatgtccagcctca agtttatatcaccacccccaatgcccaacacttgtatggccttggcgggtcatcccccccccca cccccagtatctgcaacctcaagctagcttgggtgcgttggttgtggataagtagctagactcc agcaaccagtaacctctgccctttctcctccatgacaaccagGTCCCAGGTCCCGAAAACCAAA GAAGAAGAACGCAGATCCAGAAGTTCCTATTCCGAAGTTCCTATTCTCTAGAAAGTATAGGAAC TTCTCAGATCTGGACTCTAGAGGATCCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGC GTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGG CCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCTTTGCCTGGTT TCCGGCACCAGAAGCGGTGCCGGAAAGCTGGCTGGAGTGCGATCTTCCTGAGGCCGATACTGTC GTCGTCCCCTCAAACTGGCAGATGCACGGTTACGATGCGCCCATCTACACCAACGTAACCTATC CCATTACGGTCAATCCGCCGTTTGTTCCCACGGAGAATCCGACGGGTTGTTACTCGCTCACATT
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TAATGTTGATGAAAGCTGGCTACAGGAAGGCCAGACGCGAATTATTTTTGATGGCGTTAACTCG GCGTTTCATCTGTGGTGCAACGGGCGCTGGGTCGGTTACGGCCAGGACAGTCGTTTGCCGTCTG AATTTGACCTGAGCGCATTTTTACGCGCCGGAGAAAACCGCCTCGCGGTGATGGTGCTGCGTTG GAGTGACGGCAGTTATCTGGAAGATCAGGATATGTGGCGGATGAGCGGCATTTTCCGTGACGTC TCGTTGCTGCATAAACCGACTACACAAATCAGCGATTTCCATGTTGCCACTCGCTTTAATGATG ATTTCAGCCGCGCTGTACTGGAGGCTGAAGTTCAGATGTGCGGCGAGTTGCGTGACTACCTACG GGTAACAGTTTCTTTATGGCAGGGTGAAACGCAGGTCGCCAGCGGCACCGCGCCTTTCGGCGGT GAAATTATCGATGAGCGTGGTGGTTATGCCGATCGCGTCACACTACGTCTGAACGTCGAAAACC CGAAACTGTGGAGCGCCGAAATCCCGAATCTCTATCGTGCGGTGGTTGAACTGCACACCGCCGA CGGCACGCTGATTGAAGCAGAAGCCTGCGATGTCGGTTTCCGCGAGGTGCGGATTGAAAATGGT CTGCTGCTGCTGAACGGCAAGCCGTTGCTGATTCGAGGCGTTAACCGTCACGAGCATCATCCTC TGCATGGTCAGGTCATGGATGAGCAGACGATGGTGCAGGATATCCTGCTGATGAAGCAGAACAA CTTTAACGCCGTGCGCTGTTCGCATTATCCGAACCATCCGCTGTGGTACACGCTGTGCGACCGC TACGGCCTGTATGTGGTGGATGAAGCCAATATTGAAACCCACGGCATGGTGCCAATGAATCGTC TGACCGATGATCCGCGCTGGCTACCGGCGATGAGCGAACGCGTAACGCGAATGGTGCAGCGCGA TCGTAATCACCCGAGTGTGATCATCTGGTCGCTGGGGAATGAATCAGGCCACGGCGCTAATCAC GACGCGCTGTATCGCTGGATCAAATCTGTCGATCCTTCCCGCCCGGTGCAGTATGAAGGCGGCG GAGCCGACACCACGGCCACCGATATTATTTGCCCGATGTACGCGCGCGTGGATGAAGACCAGCC CTTCCCGGCTGTGCCGAAATGGTCCATCAAAAAATGGCTTTCGCTACCTGGAGAGACGCGCCCG CTGATCCTTTGCGAATACGCCCACGCGATGGGTAACAGTCTTGGCGGTTTCGCTAAATACTGGC AGGCGTTTCGTCAGTATCCCCGTTTACAGGGCGGCTTCGTCTGGGACTGGGTGGATCAGTCGCT GATTAAATATGATGAAAACGGCAACCCGTGGTCGGCTTACGGCGGTGATTTTGGCGATACGCCG AACGATCGCCAGTTCTGTATGAACGGTCTGGTCTTTGCCGACCGCACGCCGCATCCAGCGCTGA CGGAAGCAAAACACCAGCAGCAGTTTTTCCAGTTCCGTTTATCCGGGCAAACCATCGAAGTGAC CAGCGAATACCTGTTCCGTCATAGCGATAACGAGCTCCTGCACTGGATGGTGGCGCTGGATGGT AAGCCGCTGGCAAGCGGTGAAGTGCCTCTGGATGTCGCTCCACAAGGTAAACAGTTGATTGAAC TGCCTGAACTACCGCAGCCGGAGAGCGCCGGGCAACTCTGGCTCACAGTACGCGTAGTGCAACC GAACGCGACCGCATGGTCAGAAGCCGGGCACATCAGCGCCTGGCAGCAGTGGCGTCTGGCGGAA AACCTCAGTGTGACGCTCCCCGCCGCGTCCCACGCCATCCCGCATCTGACCACCAGCGAAATGG ATTTTTGCATCGAGCTGGGTAATAAGCGTTGGCAATTTAACCGCCAGTCAGGCTTTCTTTCACA GATGTGGATTGGCGATAAAAAACAACTGCTGACGCCGCTGCGCGATCAGTTCACCCGTGCACCG CTGGATAACGACATTGGCGTAAGTGAAGCGACCCGCATTGACCCTAACGCCTGGGTCGAACGCT GGAAGGCGGCGGGCCATTACCAGGCCGAAGCAGCGTTGTTGCAGTGCACGGCAGATACACTTGC TGATGCGGTGCTGATTACGACCGCTCACGCGTGGCAGCATCAGGGGAAAACCTTATTTATCAGC
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9. Appendix
CGGAAAACCTACCGGATTGATGGTAGTGGTCAAATGGCGATTACCGTTGATGTTGAAGTGGCGA GCGATACACCGCATCCGGCGCGGATTGGCCTGAACTGCCAGCTGGCGCAGGTAGCAGAGCGGGT AAACTGGCTCGGATTAGGGCCGCAAGAAAACTATCCCGACCGCCTTACTGCCGCCTGTTTTGAC CGCTGGGATCTGCCATTGTCAGACATGTATACCCCGTACGTCTTCCCGAGCGAAAACGGTCTGC GCTGCGGGACGCGCGAATTGAATTATGGCCCACACCAGTGGCGCGGCGACTTCCAGTTCAACAT CAGCCGCTACAGTCAACAGCAACTGATGGAAACCAGCCATCGCCATCTGCTGCACGCGGAAGAA GGCACATGGCTGAATATCGACGGTTTCCATATGGGGATTGGTGGCGACGACTCCTGGAGCCCGT CAGTATCGGCGGAATTCCAGCTGAGCGCCGGTCGCTACCATTACCAGTTGGTCTGGTGTCAGGG GATCCCCCGGGCTGCAGCCAATATGGGATCGGCCATTGAACAAGATGGATTGCACGCAGGTTCT CCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTG ATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTC CGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTT CCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAG TGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGA TGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACAT CGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAG AGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGA GGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTT TCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTA CCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTAT CGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAgcggga ctctggggttcgaaatgaccgaccaagcgacgcccaacctgccatcacgagatttcgattccac cgccgccttctatgaaaggttgggcttcggaatcgttttccgggacgccggctggatgatcctc cagcgcggggatctcatgctggagttcttcgcccaccccccggatctaagctctagccggccgc ctcgagaagttcctattccgaagttcctattctctagaaagtataggaacttctaaggacgagc tgtgattgaattccgccccccccccccccccctctccctcccccccccctaacgttactggccg aagccgcttggaataaggccggtgtgcgtttgtctatatgttattttccaccatattgccgtct tttggcaatgtgagggcccggaaacctggccctgtcttcttgacgagcattcctaggggtcttt cccctctcgccaaaggaatgcaaggtctgttgaatgtcgtgaaggaagcagttcctctggaagc ttcttgaagacaaacaacgtctgtagcgaccctttgcaggcagcggaaccccccacctggcgac aggtgcctctgcggccaaaagccacgtgtataagatacacctgcaaaggcggcacaaccccagt gccacgttgtgagttggatagttgtggaaagagtcaaatggctctcctcaagcgtattcaacaa ggggctgaaggatgcccagaaggtaccccattgtatgggatctgatctggggcctcggtgcaca tgctttacgtgtgtttagtcgaggttaaaaaacgtctaggccccccgaaccacggggacgtggt
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9. Appendix tttcctttgaaaaacacgatgataatatggccacaaccatgctgctgctgctgctgctgctggg cctgaggctacagctctccctgggcatcatcccagttgaggaggagaacccggacttctggaac cgcgaggcagccgaggccctgggtgccgccaagaagctgcagcctgcacagacagccgccaaga acctcatcatcttcctgggcgatgggatgggggtgtctacggtgacagctgccaggatcctaaa agggcagaagaaggacaaactggggcctgagatacccctggccatggaccgcttcccatatgtg gctctgtccaagacatacaatgtagacaaacatgtgccagacagtggagccacagccacggcct acctgtgcggggtcaagggcaacttccagaccattggcttgagtgcagccgcccgctttaacca gtgcaacacgacacgcggcaacgaggtcatctccgtgatgaatcgggccaagaaagcagggaag tcagtgggagtggtaaccaccacacgagtgcagcacgcctcgccagccggcacctacgcccaca cggtgaaccgcaactggtactcggacgccgacgtgcctgcctcggcccgccaggaggggtgcca ggacatcgctacgcagctcatctccaacatggacattgatgtgatcctaggtggaggccgaaag tacatgtttcgcatgggaaccccagaccctgagtacccagatgactacagccaaggtgggacca ggctggacgggaagaatctggtgcaggaatggctcggcgaacgccagggtgcccggtacgtgtg gaaccgcactgagctcatgcaggcttccctggacccgtctgtgacccatctcatgggtctcttt gagcctggagacatgaaatacgagatccaccgagactccacactggacccctccctgatggaga tgacagaggctgccctgcgcctgctgagcagacacccccgcggcttcttcctcttcgtggaggg tggtcgcatcgaccatggtcatcatgaaagcagggcttaccgggcactgactgagacgatcatg ttcgacgacgccattgagagggcgggccagctcaccagcgaggaggacacgctgagcctcgtca ctgccgaccactcccacgtcttctccttcggaggctaccccctgcgagggagctccttcatcgg gctggccgctggcaaggcccgggacaggaaggcctacacggtcctcctatacggaaacggtcca ggctatgtgctcaaggacggcgcccggccggatgttaccgagagcgagagcgggagccccgagt atcggcagcagtcagcagtgcccctggacgaagagacccacgcaggcgaggacgtggcggtgtt cgcgcgcggcccgcaggcgcacctggttcacggcgtgcaggagcagaccttcatagcgcacgtc atggccttcgccgcctgcctggagccctacaccgcctgcgacctggcgccccccgccggcacca ccgacgccgcgcacccggggcggtccgtggtccccgcgttgcttcctctgctggccgggaccct gctgctgctggagacggccactgctccctgagtgtcccgtccctggggctcctgcttccccatc ccggagttctcctgctccccacctcctgtcgtcctgcctggcctccagcccgagtcgtcatccc cggagtccctatacagaggtcctgccatggaaccttcccctccccgtgcgctctggggactgag cccatgacaccaaacctgccccttggctgctctcggactccctaccccaaccccagggactgca ggttgtgccctgtggctgcctgcaccccaggaaaggagggggctcaggccatccagccaccacc tacagcccagtgggtcgctctagagcgacctcgaggggctaganntgatcataatcagccatac cacatttgtagaggttttacttgctttaaaaaacctcccacacctccccctgaacctgaaacat aaaatgaatgcaattgttgttgttaacttgtttattgcagcttataatggttacaaataaagca atagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaa
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9. Appendix actcatcaatgtatcttatcatgtctggatccccgggcgagctcgaattcgtaatcatggtcat agctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcat aaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactg cccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcgggga gaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgt tcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggg gataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccg cgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaag tcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctc gtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaa gcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaa gctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgt cttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggatta gcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacac tagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggt agctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcaga ttacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctca gtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctag atccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctg acagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccat agttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagt gctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccag ccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattg ttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgct acaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgat caaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgat cgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattct cttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattct gagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgcc acatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaagg atcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcat cttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaaggg aataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatt tatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaatag
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9. Appendix gggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgac attaacctataaaaataggcgtatcacgaggccctttcgtctcgcgcgtttcggtgatgacggt gaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccggga gcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgc ggcatcagagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgta aggagaaaataccgcatcaggcgccattcgccattcaggctgcgcaactgttgggaagggcgat cggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaag ttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgccaagctgcgt cgacAAGGGAGTGGGGACGGGCTTTTAGTTCTACTTGTCATAGTGTCTGGAAGCCAAGGTCAGG AATGGATCAACTATGCCCAGTCACAGGAGAGTGGCTGGTAGGAGCCAAGTATCTATCTTCCACC ATGGCCCAGTACTGTTTTTACATTTAGAAAACCAGCTTGTCTTTCCAAGGCCTTGCTGCTGGGC CTCTCCAACCATTGAGGTAACTACTTCATTCACATTGAGACATTCTGTTCATTCTCATGTCTAA GACAACTCAAGACTGGAAAGCCAGGAGTGATGGTTCATATTCTATAATCCCAGCACTTGAAATG TAAAGTCTAAAAACCAAGCCAGTGTGTGGGGGTGTGGTGGGGGCAGAGACAGAGAGATGAGATA TGGCTCAAAGGTTAAAAGCACCTACTACTACTACTAGCACCCAGCTAAGATGGCTCACAACCAC CCACCTATAAGCTCCAAGGGATCCAGTGCCCTCTGCTGCCTCCATGGGCACCTACACTCAAATG CACATTATTAAAAATGAAATAAACTAGGCCCATGGAGGCCAGAAAAGAGCATCAGCTCCCCCCA GACCTGGAGTTATAGGCAGTCGAAGCTGTCTGTAGGTGGTGAGAATCTGGGCATTCAAGTCTGC TCACCCCGGGGCCCCTTCCCGCCCATACCTTTAAT
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9. Appendix
9.2. EVH1‐β‐geo Fusion Protein
Murine Spred2 Gene Containing the pGT0 Gene Trap Vector behind Exon 4 and Appropriate Primers Used for Amplification of the EVH1--geo Fusion Protein and the -geo Reporter Protein
Sequence Origin: ENSEMBL Database Chromosome 11: 19,924,442 - 20,022,577, Corresponding mRNA
Cloned EVH1-β-geo Fusion Protein (4,422 bp): 1 bp – 4,422 bp (Pr.4 – Pr.6)
Cloned β-geo Reporter Protein (3,984 bp): 439 bp - 4,422 bp (Pr.5 – Pr.6)
Legend: Spred2 - Exon 1 Spred2 - Exon 2 Spred2 - Exon 3 Spred2 - Exon 4 -geo CDS Primer S2/EVH1-β-geo -Forward, Pr. 4 Primer β-geo-Forward, Pr. 5: Primer Reverse (for both), Pr. 6:
ATGACCGAAGAAACACACCCGGACGATGACAGCTATATTGTGCGTGTCAAGGCTGTGGTTATGA CCAGAGATGACTCCAGCGGGGGATGGTTCCCACAGGAAGGAGGCGGGATCAGTCGCGTCGGCGT GTGTAAGGTCATGCACCCTGAAGGCAACGGACGAAGCGGCTTTCTCATCCATGGCGAGCGACAG AAAGACAAACTGGTGGTATTGGAATGCTATGTCAGAAAGGACTTGGTCTACACCAAAGCCAATC CGACGTTTCATCATTGGAAGGTTGATAACAGGAAGTTTGGACTTACTTTCCAAAGTCCTGCAGA TGCACGAGCCTTTGACAGGGGCGTGAGAAAAGCCATTGAAGACCTTATAGAAGGTTCAACGACC TCCTCTTCCACTCTCCATAACGAAGCTGAGCTCGGAGACGATGACGTTTTCACGGTCCCAGGTC CCGAAAACCAAAGAAGAAGAACGCAGATCCAGAAGTTCCTATTCCGAAGTTCCTATTCTCTAGA
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9. Appendix
AAGTATAGGAACTTCTCAGATCTGGACTCTAGAGGATCCCGTCGTTTTACAACGTCGTGACTGG GAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTA ATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCG CTTTGCCTGGTTTCCGGCACCAGAAGCGGTGCCGGAAAGCTGGCTGGAGTGCGATCTTCCTGAG GCCGATACTGTCGTCGTCCCCTCAAACTGGCAGATGCACGGTTACGATGCGCCCATCTACACCA ACGTAACCTATCCCATTACGGTCAATCCGCCGTTTGTTCCCACGGAGAATCCGACGGGTTGTTA CTCGCTCACATTTAATGTTGATGAAAGCTGGCTACAGGAAGGCCAGACGCGAATTATTTTTGAT GGCGTTAACTCGGCGTTTCATCTGTGGTGCAACGGGCGCTGGGTCGGTTACGGCCAGGACAGTC GTTTGCCGTCTGAATTTGACCTGAGCGCATTTTTACGCGCCGGAGAAAACCGCCTCGCGGTGAT GGTGCTGCGTTGGAGTGACGGCAGTTATCTGGAAGATCAGGATATGTGGCGGATGAGCGGCATT TTCCGTGACGTCTCGTTGCTGCATAAACCGACTACACAAATCAGCGATTTCCATGTTGCCACTC GCTTTAATGATGATTTCAGCCGCGCTGTACTGGAGGCTGAAGTTCAGATGTGCGGCGAGTTGCG TGACTACCTACGGGTAACAGTTTCTTTATGGCAGGGTGAAACGCAGGTCGCCAGCGGCACCGCG CCTTTCGGCGGTGAAATTATCGATGAGCGTGGTGGTTATGCCGATCGCGTCACACTACGTCTGA ACGTCGAAAACCCGAAACTGTGGAGCGCCGAAATCCCGAATCTCTATCGTGCGGTGGTTGAACT GCACACCGCCGACGGCACGCTGATTGAAGCAGAAGCCTGCGATGTCGGTTTCCGCGAGGTGCGG ATTGAAAATGGTCTGCTGCTGCTGAACGGCAAGCCGTTGCTGATTCGAGGCGTTAACCGTCACG AGCATCATCCTCTGCATGGTCAGGTCATGGATGAGCAGACGATGGTGCAGGATATCCTGCTGAT GAAGCAGAACAACTTTAACGCCGTGCGCTGTTCGCATTATCCGAACCATCCGCTGTGGTACACG CTGTGCGACCGCTACGGCCTGTATGTGGTGGATGAAGCCAATATTGAAACCCACGGCATGGTGC CAATGAATCGTCTGACCGATGATCCGCGCTGGCTACCGGCGATGAGCGAACGCGTAACGCGAAT GGTGCAGCGCGATCGTAATCACCCGAGTGTGATCATCTGGTCGCTGGGGAATGAATCAGGCCAC GGCGCTAATCACGACGCGCTGTATCGCTGGATCAAATCTGTCGATCCTTCCCGCCCGGTGCAGT ATGAAGGCGGCGGAGCCGACACCACGGCCACCGATATTATTTGCCCGATGTACGCGCGCGTGGA TGAAGACCAGCCCTTCCCGGCTGTGCCGAAATGGTCCATCAAAAAATGGCTTTCGCTACCTGGA GAGACGCGCCCGCTGATCCTTTGCGAATACGCCCACGCGATGGGTAACAGTCTTGGCGGTTTCG CTAAATACTGGCAGGCGTTTCGTCAGTATCCCCGTTTACAGGGCGGCTTCGTCTGGGACTGGGT GGATCAGTCGCTGATTAAATATGATGAAAACGGCAACCCGTGGTCGGCTTACGGCGGTGATTTT GGCGATACGCCGAACGATCGCCAGTTCTGTATGAACGGTCTGGTCTTTGCCGACCGCACGCCGC ATCCAGCGCTGACGGAAGCAAAACACCAGCAGCAGTTTTTCCAGTTCCGTTTATCCGGGCAAAC CATCGAAGTGACCAGCGAATACCTGTTCCGTCATAGCGATAACGAGCTCCTGCACTGGATGGTG GCGCTGGATGGTAAGCCGCTGGCAAGCGGTGAAGTGCCTCTGGATGTCGCTCCACAAGGTAAAC AGTTGATTGAACTGCCTGAACTACCGCAGCCGGAGAGCGCCGGGCAACTCTGGCTCACAGTACG CGTAGTGCAACCGAACGCGACCGCATGGTCAGAAGCCGGGCACATCAGCGCCTGGCAGCAGTGG
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CGTCTGGCGGAAAACCTCAGTGTGACGCTCCCCGCCGCGTCCCACGCCATCCCGCATCTGACCA CCAGCGAAATGGATTTTTGCATCGAGCTGGGTAATAAGCGTTGGCAATTTAACCGCCAGTCAGG CTTTCTTTCACAGATGTGGATTGGCGATAAAAAACAACTGCTGACGCCGCTGCGCGATCAGTTC ACCCGTGCACCGCTGGATAACGACATTGGCGTAAGTGAAGCGACCCGCATTGACCCTAACGCCT GGGTCGAACGCTGGAAGGCGGCGGGCCATTACCAGGCCGAAGCAGCGTTGTTGCAGTGCACGGC AGATACACTTGCTGATGCGGTGCTGATTACGACCGCTCACGCGTGGCAGCATCAGGGGAAAACC TTATTTATCAGCCGGAAAACCTACCGGATTGATGGTAGTGGTCAAATGGCGATTACCGTTGATG TTGAAGTGGCGAGCGATACACCGCATCCGGCGCGGATTGGCCTGAACTGCCAGCTGGCGCAGGT AGCAGAGCGGGTAAACTGGCTCGGATTAGGGCCGCAAGAAAACTATCCCGACCGCCTTACTGCC GCCTGTTTTGACCGCTGGGATCTGCCATTGTCAGACATGTATACCCCGTACGTCTTCCCGAGCG AAAACGGTCTGCGCTGCGGGACGCGCGAATTGAATTATGGCCCACACCAGTGGCGCGGCGACTT CCAGTTCAACATCAGCCGCTACAGTCAACAGCAACTGATGGAAACCAGCCATCGCCATCTGCTG CACGCGGAAGAAGGCACATGGCTGAATATCGACGGTTTCCATATGGGGATTGGTGGCGACGACT CCTGGAGCCCGTCAGTATCGGCGGAATTCCAGCTGAGCGCCGGTCGCTACCATTACCAGTTGGT CTGGTGTCAGGGGATCCCCCGGGCTGCAGCCAATATGGGATCGGCCATTGAACAAGATGGATTG CACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAA TCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAA GACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCC ACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGC TATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATC CATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCAC CAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATG ATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCAT GCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAA AATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACA TAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGT GCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTC TTCTGA
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9.3. CRH Promoter Reporter Assay
Murine CRH Promoter Region and Appropriate Primers Used for Amplification of the CRH Promoter Region Including the RE-1/NRSE Regulatory Element
Generation of CRH Promoter Region Lacking the RE-1/NRSE Regulatory Element by Eco91I Restriction Endonuclease Digest
Sequence Origin: ENSEMBL Database Chromosome 3:19,593,401 - 19,595,396, Genomic DNA
Illustrated and Analyzed CRH Promoter Region: 6,996 bp (until end of Exon 2)
Cloned CRH Promoter Region Including RE-1/NRSE (5,834 bp): 42 bp – 5,875 bp (Pr.7 – Pr.8)
Cloned CRH Promoter Region Lacking RE-1/NRSE (5,669 bp): 42 bp – 5,710 bp (Pr.7 – Eco91I Site)
Legend: CRH-Exon 1/2 Primer CRH-RE-1/NRSE-Forward, Pr. 7 Primer CRH-RE-1/NRSE-Reverse, Pr. 8 REGULATORY ELEMENT (RE-1/NRSE) G/GTCACC : Eco91I Site
ACTTCTAATTCCCAGGATCTTAGAAGGAACTGATTTGAGAAATGGCCTTTCCAAGGGTAATTCA GTAAAATTAGGTCAAAGGCAAATGCTTGTGCCATATTTTAAGCGGTTGGGAGTAATAGCTGTGG ATTTGCTCTAAGGATTGTATTATGTTAATTGGGTTCCAAAATTTGCATGAGAATCCGCAGCATG TAAGACTCTGAGGGTCCCTGCACACAGTTGGTTTGGATTGGTAAATAAAGTTGCTGGAGGCCAA TGGCTGGGCAGGGAGGGACACAGAGGCAAGACTTTAGGATTCCCTGGCAAGGGAACAGAGAGAG GATGGAGGAGAAAGATTTGCCCTGGAGGGAAAGGAGAAAGACCAGGCCTGAGAAGTGTAGGACA
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9. Appendix
GAAAGACTGTTAACATGTAAGAGTCAGGTATCCTGGCCCAGAAGGGTTGCAAGCCTAGGGTATG GGGTGGCTAGGATGGAATATAGGTTTTAGTAAGTAATAATTCAGGAATATCATAGGGGAGAGTG TGCTAACTTCATGGAAGTTCGGAGGTGTCCAGCCATTGAGCTCTTTAAGGCATATTAAAACAAA AGTCTGTGTGTGTGTCTTTTATTCAGGAACCCAGAACACTGGGGTAGGTAGAGAGGACAAGGCC AGGCAATTTGAGCAAGAGTAACTGGCCTACCACAACAGGAAATAATAGAATACAGTTGTGCTAT TTAGTTATTTTATGATGATATAGTTCTAGATGAATTTTCTGTCCTGCGTTATTCAATGAACTAT GGAGTATCCACTGCACTTGCTTAGCTTTTGGAGTGACCACTGCATTAATTCAACAGGCAAGAAA TACGAGCTGTACTCAACAGGCTAAGCAATGTCTACTACATTTATTCAAAAGTCTGGGGAGTAGC CACTGCACATATTCAACAGTCTTGGGAATATCCACTGCATTTATTCAACAGGATTTAGAGTGTC CACTGCACTCATTAAACAGAAGAGTGACCACTACATTCTTTCAATAGGCTAGCAAGTATCCACT GAATGGACTCAACAGACAAGGGAGTAGCTTCTGGATTTATTTAACAGGCCAAGGAATGAATATC CATTGTGTTTTGAGAAGGAACTTATACAGAAAACCCATGCCCTGTCACACAGTGTCCAGTCTAT GCTGTGCCAGTGACTAGTGAAATGTGAGATTTATGCTGAAAGACATCCATGATTTTAGCAGAAC ATAAGCATACAGGTCCTAAACTTAATCTCAGAAAGAGTCCCACAACAAAACACTTGTTAATGAA ATAATTAATGTGCATTGCCAATAACACACACAGCATGCAACATATTTTGATATATATATAATAG AACATGATAGAACATCTAAGTGGAACCAACTAACCTGTATGTCACCTTACATACCATTTCCTAT GGTAAGAATATTTAAAATCACTCCCTCCCTCTTTTTTTCTTTACATTTAAGTTTTAAAATGTAG GTTTTATTTTAAAGTAGAGATCACAGTTAAGACCACAGTATTAGTCCAATTAAGAAAAATATAT GATCTCATCACAAACCTTTGGGGACAGGTAACAAAGTGGTAAATGTGATTAGCTTTGGCATTCA GAAGAATTTTGTACAGTGTTGTTTGGCCAAGTCTTACAATTTATGCCAGGCTGACTTCCAGCTT GCAGTTCTGCTGTCCACTGAAGGTGCTGCGATTAGAGATGTGTGCCTCCACAGCTGGCTTACAT GCTCCATTGTGTTTTTCAGAATCTTCCATGTTCTCTATAGAGAGGATGTATGGCTTTGACACCA GAAGAGATTGCTAGCAGTTACAATATTGTTCCCAGGAAAGCAGTAAGGTATTTATGTTTTTGAA ATCCTTTCTTTATCATCTTATGATGATGGCTCTCAAATCTACCTAAAGTGCAGTGACCACAATG ATTAAGCAACCTGACTGTGAGTAAACATGACAGATTAATGTCAACTCAGCAAACTTGGAGAGTG TATGATGGCCTGGATTTTGCCAATAGGAGTATATAAAAGTGCAAAGAGATGCAGGCTCTGAGGA GGTACCTGCTCTCTGTCTCTTTGTGTCTTGTTCTTAAAATCTGTCTCTGTCTCTGTCTCTCTCT GTCTCTCTGTCTCTCTGTCTCTGTCTCTGTCTCTCTCTCTCTCTCTCTCTCTCTCTGTGTATGT GTGTGCCTATGTGTCTATGTGCCTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGT GTGTGTGTGAGGTACTATGGCTTGAACCCAGGGCAAGTTCTTTGTCACTGAGGCCCATACCCTA CCCTCTTGTTTCTCTTTTATTTAATTAATCTGTGTTCCATATTATTTAACTCACTCCTATGTTA TATTGCTTTTGATAAAGTATTTTGAACAGGAATTAATCACTACTTGGTGCCCATATTTCTTGAA CAGTGTTTCTAGGATATAAAAGCATTTGTGTTGGTCCTTTTATGATTCCTTTCAGTAAGTGAAG TGCTATCTCTTGGTGTGAGTCTTGTAATAGAGATTATTAGTGTCTTGTGTAGATCATATCTACA
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9. Appendix
TTGTTATGTCTGTAATCACACATCAATGACATAATTTCAAGAATCCTCCCTACTCCCTATATTC TATAAAAAGATAAAATTAATTCTTTTATTTTTGTGTGAATTTTTATTTTGCATTTTCATTTTTA GGTGCATCAGTGCACTGAAAAACCTCACATCACAAGATTCAGCTAACATGAAAATAGCAGTGTG CACATACAGACATTTAAAGCCAAGGGGCACTGCACAAAGAATTTCTGTGTTCTTGTGCCTTGGG AATAAAAGGAAACCCCACGAAAAGGCCTCCAAGAGCTGATATGTGTGTTGCTCCACCTCGTCCA CTCGTCTCTGCTCATTCGTGTGCCTGTGCTGGGAAAGAAAGCACAAAGGATGCCGTGATGCTGT GTAAATAGTGAGGCCAGCAGTAAAGAACTGGGTTCTTGTTCTGACTTTATCACTCGCTGAGTAA CTTCAGATTCCCTCTGGTTCTCATTTAAGCCAAGCGAGCATTTCTAACACGAGTTCTCTGAGAT TTGCTTCAGTAAATTATGGTACTTCACAATTTATATTCCAGGTAAGCCCCGTGGCAAAGGCTCA ACAGCAATAGTGGAGCCCGAGTAAAAACACGTCTCCTGATGGTCCGATGGGAGGGATCTTGAGG GCCAGGAGGTTAGCAAGAGCCCATGTGTGCCACTTCTTCCCCCAGGGACCCTCCCTGACTTCTC CTGGAAAGCCCATAGGCATGTCTCTCTCTTACTTCCTCTTCAGGACTCATTGGCCTCCTTTCCC TCTCCAGACTGGCACTTAAGCTGAACTTTATGAATGGGCAAAGAATTGGTCAGGAATGAAAGTT AAAACAAAACAGAGACCAACAAACAAAAATCCCAAACACTGAGGTGAATTTTAACCTCTAAAAT GCCCACTGTAACAATAGTGAAGGTTCTGCAGGCCTCCTGGTCAATGTAAACCCACTTTTCCTCT AAAGCACAATTTGATCATTATTTATTAATGAATCACACTTCTCCTGTGGACAGAATCCTTCCTG TGTTCTCTTCTATCAGAAATGCATCCCATTCATTCACCTATGAGCTGACTTTTCTGCAACCTCC AGTGTGAAAACACCTTCCTCTTGTTTCAAGAAGAGTTATCAATTAGTACAGGCCCCAAACAGGG GTGGAAATCTGCATTGCATTACTTGTGACAGAGAAAATGGCAGACATAACCAGAAACAGGGAAC AGGAAATTTTGGATAAACAGTAGTGGTGGTTATGAAGCAGCTGAAGCGAATCCCAGACACTAGA GTTTGTAACAGCATCTTGAATTGCAGGATTAAAGGTAACTACTTCCCACTTAGACATAATCTCC ATTCAACTCAAAATTTGAGAGTAGATTTATAGACATGGCATTGTGTTCTATAATAGTGCCAGAA AGCAATAACTAAAGACACTTGTGGTGCACATTTGAATGTGGAGAAAAGAACAGAACACATAATA AATAACTTGAGTGGTTTGCTCTGCATACCATCATGGTTTCACAGTACACGGAGGTGAAATGAAT GATTCTCATTTACAGACTCAATAACTGATTTTTTTTCAGGCTATCAAACAGCTTAAGTTTTTTC AACACAAGAGAACAATGCCTCTCTCATTGGGTTTGCGGTGTAGACTCCGAACAGAGTGTTTAAT ATACACAGCTCACCCTGCTCTTCTTGGGGGAACAGTCCTGATTAACTTTAGATTCTAAGAGAAC CATAGGAGGCCTGAGATTCACTTGAAACTGAGGCTTTTAAGAGACTTTGTTTATGGCCTTCCTC GTTAGAATAGCTCTTTTCTTGTGAAGGAAAACACTAAGCTGGTTTCAAAAAAACGTACTGGCCT TGTCTTCAGCTCCATAATTCCTATGTATGCTCTTGGGAGGGCTAAGTGGATAAAAACTCAGACC TGGTTGTCTCTGTCCCCTAGTTCTTCATTCTCCTCCAGGCAGAAAGATGGCAGGCCATAGTAAC GACAGATTTCAGATACTGAGATGTTTCCTGAGAGGACAACCTGACAGAAGAGTTAGGTGGGGGT GCTGAGGCACCAGGAAATGTCCAGATCCACCCCTTTAATGGTAACTACCTTCTTGGAAGCTCTG CACTCCCCACCTCTTCTTCTTTCCCCTCCCACTCTCTGTCTCTTTTCTGGTCCGTATCTGGCCT
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ATCATAGTAAGAGGTCAGTCTGTTTTCCACACTTGGATAGTCTCATTCAAAAATTTTTGTCAAT GGACAAGTCATAAGAAACCCTTCCATTTTCGGGCTCGTTGACGTCACCAAGGAGGCGATAAATA TCTGTTGATATAATTGGATGTGAGATTCAGTGTTGAAATAGCAGAACTCTGTCCCTCGCTCCTT GGCAGGGCCCTATTATTTATGCAGGAGCAGAGGCAGCACGCAATCGAGCTGTCAAGAGAGCGTC AGCTTATTAGGCAAATGCTGCGTGCTTTCTGAAGAGGGTCGACATTATAAAATCTCACTCCAGG CTCTGGTGTGGAGAAACTCAGAGCCCAAGTACGTTGAGAGACTGAAGAGAAAGGGAAAAGGCAA AAGAAAAAAAGAAGAGAAAGGAGAAGAGGAAGAAAACCTGCAGGAGGCATCCTGAGAGAAGTCC CTCTGCAGAGGCAGCAGTGCGGGCTCACCTACCAAGGGAGGAGAAGGTAGGCAGCGCCTAGACG GGCGCCACCAACTTTGTGCTGCCTGAGCTGCTGTGGTGAGCCCCGGAGCCAGCTGCCCATGTGC TGGAATGCCTGTGCCTATGCATGTATGTGTGTCGCTAACTGTGCCTTAAAATTCCGATGACAGT GGCGATTTGAAAAAGCGAAGTTAGACGGCGGCTGCTCATCTTTATCCACTCAATCCAATCTGCC ACTCACTGCTCATAGTCTGTGCAAAGAATGGCTCCCCTATTGCATCCCATGTCCCCAAGCAAAC GGAGTAAGGGCAGGAATGGAGACAGAGAAGGTTGTTCTCAATTTGGCAGAAAAGGATGTCCGAA AGGGGGCGATTAGGGTGTGCGACAGCTTAAACCTGTGGCACTTGTCCGGGCTCAGGGAAGTCGG TTTAGGGAAGACGTTTGGGAGGTCCTTAGGAAGAGGAGCCAGGGGTTGTCCCTTCTAGGTCTCC AAAGAAGGGTCACCGCGGGCTCGCACCAGTTGAGCTTTGCAGGTACCTAGCTTCAGCACCGCGG ACAGCGTCACCGAAGCCTAGAGCCTGTCTTGTCTGTGGGTGTCCGATAGGAAGCCCCGCTGCAC CTTCCAGCTGAGCTAAACTCTGACCAATCTTACCTTTCTCCCCCACCTTCTCTCTCCCCCGACC TCAACCTCGGTGCTTCAGAGAGCGCCCCTAACATGCGGCTGCGGCTGCTGGTGTCCGCGGGCAT GCTGCTGGTGGCTCTGTCGTCCTGCCTGCCTTGCAGGGCCCTGCTCAGCAGGGGATCCGTCCCC CGAGCGCCGCGGGCCCCGCAGCCCTTGAATTTCTTGCAGCCGGAGCAGCCCCAGCAACCTCAGC CGGTTCTGATCCGCATGGGTGAAGAATACTTCCTCCGCCTGGGGAATCTCAACAGAAGTCCCGC TGCTCGGCTGTCCCCCAACTCCACGCCCCTCACCGCGGGTCGCGGCAGCCGCCCCTCGCACGAC CAGGCTGCGGCTAACTTTTTCCGCGTGTTGCTGCAGCAGCTGCAGATGCCTCAGCGCTCGCTCG ACAGCCGCGCGGAGCCGGCCGAACGCGGCGCCGAGGATGCCCTCGGTGGCCACCAGGGGGCGCT GGAGAGGGAGAGGCGGTCGGAGGAGCCGCCCATCTCTCTGGATCTCACCTTCCACCTTCTGCGG GAAGTCTTGGAAATGGCCCGGGCAGAGCAGTTAGCTCAGCAAGCTCACAGCAACAGGAAACTGA TGGAGATTATCGGGAAATGAAATGTTGCGCTTGGCCAAAACGATTCTGCATTTAGCACACAAGT AAAAATAAAAATTTAAAACACAGTATTCTGTACCATATCGCAGCTCCGATATCATTTGTTTATT TTTATATAGCTTGAAGCATAGAAGATGTACAGGGAGAGAGCCTATATACCCCTTAATTAGCATG CACAAAGTGTATTTCTGTGTCGTAACAAAACAGCGTTATTTGTATTGCCCATGCTTAATTTCTA TGTGCAAATAAGCGTCTTTATAGCGATATCTTAAAGAAAATGTGGCCCCAAGGAGGAAACCTTT GAAAAAGCAGATGGGAGTCATCCAGTTGTTTTTATTTGGAGCTGCAGTGGAAGAGAATTCATTC TTGAGGGGTGGCTAGGACGAAATGTGTAAGCTCTTTGAATCAACTTTTTCTTGTAAATGTTTCA
171
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GTAATAAAACATCTTTCTGATTCTTGGTCAATTTGGTTGTGTAAGAGAACGTTAAATATATTTT TAATAAAATCTGCAAAGGTT
172
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9.4. Vector Cards
Plasmids with pcDNA3.1/myc-His(-) A Vector Backbone
(Invitrogen, Cat. No. V855-20)
pcDNA3.1-SPRED1 and pcDNA3.1-SPRED2
Full-length cDNAs of murine Spred1 and Spred2 including their endogenous stop codons were cloned into the NotI and HindIII sites of the pcDNA3.1/myc-His(-) A vector. With these constructs, SPRED1 and SPRED2 are expressed without myc-His tags (Bundschu, 2005).
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Plasmids with pcDNA3.1/myc-His(-) B Vector Backbone
(Invitrogen, Cat. No. V855-20)
pcDNA3.1-EVH1-β-geo and pcDNA3.1-β-geo
Full-length cDNAs of EVH1-β-geo and β-geo including their endogenous stop codons were cloned into the NotI and HindIII sites of the pcDNA3.1/myc-His(-) B vector. With these constructs, EVH1-β-geo and β-geo are expressed without myc-His tags (4.3.1./5.3.2/Appendix 9.2.)
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9. Appendix
Plasmids with pGL3 Basic Vector Backbone
(Promega, Cat. No. E1751)
pGL3-CRHProm-RE and pGL3-CRHProm
A 5.7 kb region of the murine CRH promoter region containing exon 1 was cloned into the XhoI site of the pGL3 Basic Vector. pGL3-CRHProm-RE includes the RE-1/NRSE regulatory element in the intron between CRH exons 1 and 2, pGL3-CRHProm lacks it (4.3.2./5.14.2/Appendix 9.3.). The activity of the cloned promoter sequence can be monitored because it drives the expression of the downstream firefly luciferase reporter gene. 175
9. Appendix
Plasmids with pGL2 Basic Vector Backbone
(Promega, Cat. No. E1641)
pGL2-4xEts1
4 Ets1 binding sites were cloned into the SmaI site of the pGL2 Basic Vector by blunt end ligation. Ets1 factor-dependent transcription can be monitored by firefly luciferase reporter gene expression (Appendix 9.5.).
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pRL-TK
(Promega, Cat. No. E2241)
pRL-TK contains a Renilla luciferase reporter gene, whose expression is driven by the constitutive active HSV-TK promoter. It was used as an internal control reporter plasmid in combination with the CRH promoter and 4xEts1 firefly luciferase reporter vectors.
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9.5. 4xEts1 Reporter Assay
Sequence of and Transcription Factor Binding Site Prediction in the 4xEts1 Reporter
Sequence Origin: Eurofins MWG Operon Sequence Analysis of the 4xEts1 Promoter Reporter (pGL2-4xETS1)
Sequence of 4xETS1-Insert: CCCCTAGAGCTCGACTGTGCTCAGTTAGTCACTTCCTCGACTGTGCTCAGTTAGTCACTTCCTC GACTGTGCTCAGTTAGTCACTTCCTCGACTGTGCTCAGTTAGTCACTTCCTCGAGGATCCAAGC TAGCTTGTGACCTCACCCTGCAGAGCCACACCCTGGTGTTGGCCAATCTACACACGGGGTAGGG ATTACATAGTTCAGGACTTGGGCATAAAAGGCAGAGCAGGGCAGCTGCTGCTTACACTTGCTTA GATCTAAGTAAGCTGGCATCCGTACCGTA
The Insert Contains 4 Ets1 Binding Sites: 1. 28 bp – 37 bp 2. 54 bp – 63 bp 3. 80 bp – 89 bp 4. 106 bp – 115 bp Legend: c-Ets-1 Binding Site
AliBaba2.1 predicts the following sites in your sequence:
======seq( 0.. 59) cccctagagctcgactgtgctcagttagtcacttcctcgactgtgctcagttagtcactt Segments: 1.1.1.1 21 30 ====JunD== 1.1.1.5 23 32 ====GCN4== 9.9.29 24 33 ====AP-1== 9.9.32 24 33 ====AP-1== 2.3.1.0 27 36 ====Sp1=== 3.5.2.0 28 37 ==c-Ets-1= 1.1.1.1 47 56 ====JunD==
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1.1.1.5 49 58 ====GCN4== 9.9.29 50 59 ====AP-1== 9.9.32 50 59 ====AP-1== 2.3.1.0 53 62 ====Sp1 3.5.2.0 54 63 ==c-Et ======seq( 60.. 119) cctcgactgtgctcagttagtcacttcctcgactgtgctcagttagtcacttcctcgagg Segments: 2.3.1.0 53 62 === 3.5.2.0 54 63 s-1= 1.1.1.1 73 82 ====JunD== 1.1.1.5 75 84 ====GCN4== 9.9.29 76 85 ====AP-1== 9.9.32 76 85 ====AP-1== 2.3.1.0 79 88 ====Sp1=== 3.5.2.0 80 89 ==c-Ets-1= 1.1.1.1 99 108 ====JunD== 1.1.1.5 101 110 ====GCN4== 9.9.29 102 111 ====AP-1== 9.9.32 102 111 ====AP-1== 2.3.1.0 105 114 ====Sp1=== 3.5.2.0 106 115 ==c-Ets-1======seq( 120.. 179) atccaagctagcttgtgacctcaccctgcagagccacaccctggtgttggccaatctaca Segments: 2.1.2.1 131 140 =RAR-alph= 9.9.45 131 140 ===ARP-1== 2.1.2.2 133 142 ==RXR-beta 2.1.2.3 133 142 =REV-ErbA= 2.1.1.4 133 144 ======ER==== 1.1.1.6 134 143 ==CRE-BP1= 2.3.3.0 134 143 =CPE_bind= 1.1.1.1 135 144 ===c-Jun== 1.1.3.0 135 144 =C/EBPalp= 9.9.51 135 144 ====ATF=== 9.9.77 136 145 =CACCC-bi= 2.3.1.0 137 147 =====Sp1=== 2.3.1.0 152 162 =====Sp1=== 1.1.3.0 165 174 =C/EBPalp= 9.9.539 167 176 ====NF-1== 1.3.1.2 178 187 ======seq( 180.. 239) cacggggtagggattacatagttcaggacttgggcataaaaggcagagcagggcagctgc Segments: 1.3.1.2 178 187 ==USF===
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2.3.1.0 181 193 ======Sp1==== 1.1.3.0 192 201 =C/EBPalp= 4.3.2.0 207 216 ====SRF=== 2.3.2.2 211 220 =====Kr=== 1.2.1.0 233 242 =====E1 1.2.2.0 233 242 ====Myo 3.5.1.2 233 242 ===Adf- ======seq( 240.. 285) tgcttacacttgcttagatctaagtaagctggcatccgtaccgta Segments: 1.2.1.0 233 242 === 1.2.2.0 233 242 D== 3.5.1.2 233 242 1==
47 segments in this sequence identified as potential binding sites
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9.6. Transcription Factor Binding Site Prediction in the Murine CRH Promoter
Focus on Predicted Ets1 Binding Sites
Sequence Origin: ENSEMBL Database Chromosome 3:19,593,401 - 19,595,396, Genomic DNA
Illustrated and Analyzed CRH Promoter Region: 5,906 bp (until end of CRH-Intron 1)
Cloned CRH Promoter Region Including RE-1/NRSE (5,834 bp): 42 bp – 5,875 bp (Pr.7 – Pr.8)
Contains 4 Ets1 Binding Sites: 1. 673 bp – 682 bp 2. 3576 bp – 3585 bp 3. 3772 bp – 3783 bp 4. 4617 bp – 4626 bp
Cloned CRH Promoter Region Lacking RE-1/NRSE (5,669 bp): 42 bp – 5,710 bp (Pr.7 – Eco91I Site)
Contains 4 Ets1 Binding Sites: 1. 673 bp – 682 bp 2. 3576 bp – 3585 bp 3. 3772 bp – 3783 bp 4. 4617 bp – 4626 bp
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Legend: c-Ets-1 Binding Site CRH-Exon 1/2 Primer CRH-RE-1/NRSE-Forward, Pr. 7 Primer CRH-RE-1/NRSE-Reverse, Pr. 8 REGULATORY ELEMENT (RE-1/NRSE) G/GTCACC : Eco91I Site
AliBaba2.1 predicts the following sites in your sequence:
======seq( 0.. 59) acttctaattcccaggatcttagaaggaactgatttgagaaatggcctttccaagggtaa Segments: 3.5.2.0 23 32 ====PU.1== 1.1.3.0 32 41 =C/EBPalp= 3.1.2.2 34 43 ===Oct-1== 9.9.590 42 51 =NF-kappaB ======seq( 60.. 119) ttcagtaaaattaggtcaaaggcaaatgcttgtgccatattttaagcggttgggagtaat Segments: 4.3.2.0 68 77 ====SRF=== 2.1.1.4 70 79 =====ER=== 2.1.2.1 72 81 =RAR-alph= 2.1.2.3 72 81 =REV-ErbA= 2.1.2.10 72 81 ====COUP== 4.3.1.1 97 106 ===MEB-1== 4.3.1.2 97 106 ====GLO======seq( 120.. 179) agctgtggatttgctctaaggattgtattatgttaattgggttccaaaatttgcatgaga Segments: 1.1.3.0 125 134 =C/EBPalp= 9.9.539 160 169 ====NF-1== 1.1.3.0 168 177 =C/EBPalp= 3.1.2.2 168 177 ===Oct-1======seq( 180.. 239) atccgcagcatgtaagactctgagggtccctgcacacagttggtttggattggtaaataa Segments: 1.1.3.0 227 236 =C/EBPalp= 3.1.1.12 229 238 ===HNF-1== 3.1.2.2 229 238 ===Oct-1== 4.5.1.0 231 240 ====TBP== 1.1.3.0 234 243 =C/EBP ======seq( 240.. 299) agttgctggaggccaatggctgggcagggagggacacagaggcaagactttaggattccc Segments: 4.5.1.0 231 240 = 1.1.3.0 234 243 alp= 2.3.1.0 243 252 ====Sp1=== 9.9.539 248 257 ====NF-1== 9.9.150 250 259 ====CP1=== 2.3.1.0 257 268 =====Sp1==== 2.3.1.0 263 274 =====Sp1==== 2.1.1.1 271 280 =====GR=== 1.1.3.0 279 288 =C/EBPgam= 9.9.539 295 304 ====N 9.9.535 298 307 == 182
9. Appendix
2.3.1.0 299 308 ======seq( 300.. 359) tggcaagggaacagagagaggatggaggagaaagatttgccctggagggaaaggagaaag Segments: 9.9.539 295 304 F-1== 9.9.535 298 307 ==NF-1== 2.3.1.0 299 308 ===Sp1=== 2.3.1.0 320 329 ====Sp1======seq( 360.. 419) accaggcctgagaagtgtaggacagaaagactgttaacatgtaagagtcaggtatcctgg Segments: 2.1.1.2 385 394 =====PR=== 1.1.1.5 400 409 ====GCN4== 9.9.32 401 410 ====AP-1======seq( 420.. 479) cccagaagggttgcaagcctagggtatggggtggctaggatggaatataggttttagtaa Segments: 9.9.726 449 458 =represso= 3.1.1.2 479 488 ======seq( 480.. 539) gtaataattcaggaatatcataggggagagtgtgctaacttcatggaagttcggaggtgt Segments: 3.1.1.2 479 488 ==Zen-1== 2.3.1.0 500 509 ====Sp1=== 1.1.3.0 504 513 =C/EBPbeta ======seq( 540.. 599) ccagccattgagctctttaaggcatattaaaacaaaagtctgtgtgtgtgtcttttattc Segments: 3.3.2.0 566 575 ===HNF-3== 3.5.1.2 577 589 =====RAP1==== 3.3.2.0 591 600 ===HNF-3= 3.1.2.1 593 602 ===Pit- 9.9.29 594 603 ====AP ======seq( 600.. 659) aggaacccagaacactggggtaggtagagaggacaaggccaggcaatttgagcaagagta Segments: 3.3.2.0 591 600 = 3.1.2.1 593 602 1a= 9.9.29 594 603 -1== 9.9.150 629 638 ====CP1=== 1.1.3.0 640 654 ===C/EBPalpha== 9.9.32 654 663 ====AP ======seq( 660.. 719) actggcctaccacaacaggaaataatagaatacagttgtgctatttagttattttatgat Segments: 9.9.32 654 663 -1== 3.5.2.0 673 682 =c-Ets-1_= 9.9.590 673 682 =NF-kappaB 1.1.3.0 676 685 ===C/EBP== 2.2.1.1 676 685 ===GATA-1= 1.1.3.0 693 702 =C/EBPbeta 3.1.2.2 701 710 ===Oct-1== 1.1.3.0 701 714 ===C/EBPalpha= 3.1.1.2 705 714 ====Antp======seq( 720.. 779) gatatagttctagatgaattttctgtcctgcgttattcaatgaactatggagtatccact Segments: 1.1.3.0 748 757 =C/EBPbeta ======seq( 780.. 839) gcacttgcttagcttttggagtgaccactgcattaattcaacaggcaagaaatacgagct Segments: 2.1.2.3 801 810 =REV-ErbA======seq( 840.. 899) gtactcaacaggctaagcaatgtctactacatttattcaaaagtctggggagtagccact Segments: 1.1.3.0 840 849 =C/EBPalp= 3.1.1.2 867 876 ====Dfd=== 3.1.2.2 873 882 ===Oct-1== 183
9. Appendix
2.3.1.0 882 894 ======Sp1==== 1.1.3.0 896 905 =C/E 4.3.2.0 899 908 ======seq( 900.. 959) gcacatattcaacagtcttgggaatatccactgcatttattcaacaggatttagagtgtc Segments: 1.1.3.0 896 905 BPalp= 4.3.2.0 899 908 ===SRF=== 4.1.1.0 918 927 ===c-Rel== 9.9.590 919 928 =NF-kappaB 9.9.592 919 928 =NF-kappa= 9.9.594 919 928 ====RelA== 3.1.1.2 931 940 ====Dfd=== 3.1.2.1 933 942 ===Pit-1a======seq( 960.. 1019) cactgcactcattaaacagaagagtgaccactacattctttcaataggctagcaagtatc Segments: 3.1.1.12 967 976 ===HNF-1== 3.6.1.0 990 999 ====TEC1======seq( 1020.. 1079) cactgaatggactcaacagacaagggagtagcttctggatttatttaacaggccaaggaa Segments: 4.3.2.0 1023 1032 ====SRF=== 9.9.539 1035 1044 ====NF-1== 2.3.1.0 1043 1052 ====Sp1=== 9.9.539 1070 1079 ====NF-1== 3.6.1.0 1074 1083 ====TE ======seq( 1080.. 1139) tgaatatccattgtgttttgagaaggaacttatacagaaaacccatgccctgtcacacag Segments: 3.6.1.0 1074 1083 C1== 3.1.2.2 1082 1091 ===Oct-1== 3.5.2.0 1102 1111 ====PU.1== 3.5.1.2 1119 1128 ====RAP1== 2.3.1.0 1122 1131 ====Sp1=== 2.1.2.3 1123 1132 =T3R-alpha 2.3.1.0 1128 1137 ====Sp1======seq( 1140.. 1199) tgtccagtctatgctgtgccagtgactagtgaaatgtgagatttatgctgaaagacatcc Segments: 9.9.29 1157 1166 ====AP-1== 1.1.3.0 1169 1178 =C/EBPbeta ======seq( 1200.. 1259) atgattttagcagaacataagcatacaggtcctaaacttaatctcagaaagagtcccaca Segments: 1.1.3.0 1202 1211 =C/EBPalp= 2.1.1.1 1210 1219 =====GR=== 3.1.2.2 1216 1225 ===Oct-1== 1.1.3.0 1217 1226 =C/EBPbeta 1.1.3.0 1236 1245 =C/EBPbeta 1.1.1.5 1245 1254 ====GCN4== 1.1.3.0 1256 1265 =C/E 2.3.2.2 1258 1267 ======seq( 1260.. 1319) acaaaacacttgttaatgaaataattaatgtgcattgccaataacacacacagcatgcaa Segments: 1.1.3.0 1256 1265 BPalp= 2.3.2.2 1258 1267 =LyF-1== 3.1.2.2 1273 1282 ===Oct-1== 1.1.3.0 1275 1287 ==C/EBPalpha= 3.1.1.2 1279 1288 ===Zen-1== 3.1.2.2 1286 1295 ===Oct-1== 2.2.1.1 1287 1296 ===GATA-1= 9.9.539 1291 1303 =====NF-1==== 1.1.3.0 1294 1303 =C/EBPalp= 3.1.1.12 1294 1303 ===HNF-1== 9.9.535 1294 1303 ====NF-1== 9.9.537 1294 1303 ====NF-1== 184
9. Appendix
2.3.2.3 1302 1311 =WT1_I_-K= 4.6.1.0 1302 1311 ===Sox-2== 9.9.1840 1302 1311 ===WT1_I== 9.9.1841 1302 1311 ==WT1-del2 9.9.1842 1302 1311 =WT1_I-de= 3.1.2.2 1311 1320 ===Oct-1= 1.1.3.0 1313 1322 =C/EBPa ======seq( 1320.. 1379) catattttgatatatatataatagaacatgatagaacatctaagtggaaccaactaacct Segments: 3.1.2.2 1311 1320 = 1.1.3.0 1313 1322 lp= 1.1.3.0 1321 1330 =C/EBPalp= 4.5.1.0 1329 1341 ======TBP==== 3.1.2.1 1331 1340 ===Pit-1a= 2.1.1.1 1339 1348 =====GR======seq( 1380.. 1439) gtatgtcaccttacataccatttcctatggtaagaatatttaaaatcactccctccctct Segments: 2.3.1.0 1396 1405 ====Sp1=== 1.1.3.0 1398 1407 =C/EBPalp= 9.9.590 1399 1408 =NF-kappaB 3.1.2.2 1414 1423 ===Oct-1== 1.1.3.0 1418 1427 =C/EBPalp= 2.1.1.4 1420 1429 =====ER=== 2.3.1.0 1426 1436 =====Sp1=== 4.5.1.0 1434 1443 ====TB 9.9.820 1434 1443 ===TFI 2.3.3.0 1435 1444 =CPE_ 1.1.1.5 1438 1447 == 2.3.2.2 1439 1449 ======seq( 1440.. 1499) ttttttctttacatttaagttttaaaatgtaggttttattttaaagtagagatcacagtt Segments: 4.5.1.0 1434 1443 P=== 9.9.820 1434 1443 ID== 2.3.3.0 1435 1444 bind= 1.1.1.5 1438 1447 ==GCN4== 2.3.2.2 1439 1449 ====Hb==== 2.2.1.1 1443 1452 ===GATA-1= 3.1.2.2 1444 1457 =====Oct-1==== 3.1.1.2 1447 1456 ===Zen-1== 3.1.1.2 1472 1481 ====Antp== 3.1.2.2 1475 1484 ===Oct-1== 4.3.1.1 1475 1484 ===MEB-1== 2.1.1.4 1489 1498 =====ER======seq( 1500.. 1559) aagaccacagtattagtccaattaagaaaaatatatgatctcatcacaaacctttgggga Segments: 1.1.3.0 1513 1522 =C/EBPalp= 3.1.1.0 1518 1527 =MATalpha1 3.1.2.2 1518 1527 ===Oct-1== 4.3.1.3 1518 1527 ====MCM1== 1.1.3.0 1519 1528 =C/EBPalp= 2.3.1.0 1555 1564 ====S 2.2.1.1 1557 1566 ======seq( 1560.. 1619) caggtaacaaagtggtaaatgtgattagctttggcattcagaagaattttgtacagtgtt Segments: 2.3.1.0 1555 1564 p1=== 2.2.1.1 1557 1566 GATA-1= 2.1.1.1 1564 1573 =====GR=== 2.2.1.1 1568 1577 ===GATA-1= 1.1.3.0 1605 1614 =C/EBPalp= 2.1.1.1 1607 1616 =====GR=== 4.3.2.0 1617 1626 === 9.9.537 1619 1628 ======185
9. Appendix
seq( 1620.. 1679) gtttggccaagtcttacaatttatgccaggctgacttccagcttgcagttctgctgtcca Segments: 4.3.2.0 1617 1626 =SRF=== 9.9.537 1619 1628 ===NF-1== 9.9.539 1622 1631 ====NF-1== 3.1.2.2 1633 1642 ===Oct-1== 9.9.539 1639 1648 ====NF-1== 9.9.29 1651 1660 ====AP-1======seq( 1680.. 1739) ctgaaggtgctgcgattagagatgtgtgcctccacagctggcttacatgctccattgtgt Segments: 1.3.1.4 1710 1719 ====AP-4======seq( 1740.. 1799) ttttcagaatcttccatgttctctatagagaggatgtatggctttgacaccagaagagat Segments: 3.4.1.0 1746 1755 ====HSF=== 3.1.1.2 1777 1786 ====Antp== 1.1.3.0 1794 1803 =C/EBP ======seq( 1800.. 1859) tgctagcagttacaatattgttcccaggaaagcagtaaggtatttatgtttttgaaatcc Segments: 1.1.3.0 1794 1803 alp= 2.3.1.0 1823 1832 ====Sp1=== 9.9.539 1825 1834 ====NF-1== 1.1.3.0 1840 1849 =C/EBPalp= 1.1.3.0 1849 1858 =C/EBPgam======seq( 1860.. 1919) tttctttatcatcttatgatgatggctctcaaatctacctaaagtgcagtgaccacaatg Segments: 2.2.1.1 1861 1870 ===GATA-1= 3.5.2.0 1862 1871 ===Erg-1== 1.6.1.0 1883 1892 =AP-2alph= 3.1.2.2 1913 1922 ===Oct- 1.1.3.0 1919 1928 ======seq( 1920.. 1979) attaagcaacctgactgtgagtaaacatgacagattaatgtcaactcagcaaacttggag Segments: 3.1.2.2 1913 1922 1== 1.1.3.0 1919 1928 C/EBPalp= 1.1.3.0 1937 1946 =C/EBPalp= 3.1.1.12 1937 1946 ===HNF-1== 3.3.2.0 1937 1946 ===HNF-3== 9.9.29 1942 1951 ====AP-1======seq( 1980.. 2039) agtgtatgatggcctggattttgccaataggagtatataaaagtgcaaagagatgcaggc Segments: 1.1.3.0 1996 2008 ==C/EBPalpha= 9.9.537 1999 2008 ====NF-1== 9.9.539 2000 2009 ====NF-1== 3.1.2.2 2012 2021 ===Oct-1== 4.5.1.0 2012 2022 =====TBP=== 4.5.1.0 2013 2023 =====TBP=== 1.1.3.0 2037 2046 =C/ ======seq( 2040.. 2099) tctgaggaggtacctgctctctgtctctttgtgtcttgttcttaaaatctgtctctgtct Segments: 1.1.3.0 2037 2046 EBPalp= 2.3.1.0 2043 2052 ====Sp1=== 1.1.1.5 2073 2082 ====GCN4== 2.1.1.1 2073 2082 =====GR=== 2.1.1.2 2076 2085 =====PR======seq( 2100.. 2159) ctgtctctctctgtctctctgtctctctgtctctgtctctgtctctctctctctctctct Segments: ======seq( 2160.. 2219) ctctctctgtgtatgtgtgtgcctatgtgtctatgtgcctgtgtgtgtgtgtgtgtgtgt Segments: 3.5.1.2 2167 2178 =====RAP1=== 186
9. Appendix
3.5.1.2 2200 2209 ====RAP1== 3.5.1.2 2212 2221 ====RAP1 ======seq( 2220.. 2279) gtgtgtgtgtgtgtgtgtgtgtgtgtgtgaggtactatggcttgaacccagggcaagttc Segments: 3.5.1.2 2212 2221 == 3.5.1.2 2226 2235 ====RAP1== 3.5.1.2 2239 2248 ====RAP1== 9.9.539 2256 2265 ====NF-1======seq( 2280.. 2339) tttgtcactgaggcccataccctaccctcttgtttctcttttatttaattaatctgtgtt Segments: 2.3.1.0 2299 2308 ====Sp1=== 3.5.3.0 2311 2320 ===ICSBP== 9.9.428 2311 2320 ===ISGF-3= 1.1.1.5 2316 2325 ====GCN4== 3.3.2.0 2317 2326 ===HNF-3== 4.5.1.0 2317 2326 ====TBP=== 3.1.1.12 2319 2328 ===HNF-1== 2.3.2.2 2321 2330 =====Hb=== 3.1.2.2 2321 2330 ===Oct-1== 3.1.2.2 2338 2347 ======seq( 2340.. 2399) ccatattatttaactcactcctatgttatattgcttttgataaagtattttgaacaggaa Segments: 3.1.2.2 2338 2347 =Oct-1== 1.1.3.0 2342 2351 =C/EBPbeta 3.1.1.2 2345 2354 ====Ftz=== 3.1.1.8 2345 2354 ====Eve=== 3.1.1.2 2368 2377 ====Antp== 1.1.3.0 2394 2403 =C/EBP ======seq( 2400.. 2459) ttaatcactacttggtgcccatatttcttgaacagtgtttctaggatataaaagcatttg Segments: 1.1.3.0 2394 2403 alp= 3.1.1.12 2400 2409 ===HNF-1C= 1.2.8.0 2401 2410 ====Id3=== 4.1.3.0 2401 2410 ==NF-ATc3= 1.1.3.0 2415 2424 =C/EBPbeta 4.3.2.0 2415 2424 ====SRF=== 9.9.539 2415 2424 ====NF-1== 4.5.1.0 2443 2452 ====TBP======seq( 2460.. 2519) tgttggtccttttatgattcctttcagtaagtgaagtgctatctcttggtgtgagtcttg Segments: 4.3.2.0 2466 2475 ====SRF=== 4.5.1.0 2469 2478 ====TBP=== 3.1.2.1 2470 2479 ===Pit-1a= 2.2.1.1 2494 2503 ===GATA-1= 1.1.3.0 2513 2523 =C/EBPa 1.1.3.0 2514 2523 =C/EBP ======seq( 2520.. 2579) taatagagattattagtgtcttgtgtagatcatatctacattgttatgtctgtaatcaca Segments: 1.1.3.0 2513 2523 lpha 1.1.3.0 2514 2523 alp= 2.2.1.1 2524 2533 ===GATA-1= 3.1.1.12 2543 2552 ===HNF-1== 3.1.2.2 2551 2560 ===Oct-1======seq( 2580.. 2639) catcaatgacataatttcaagaatcctccctactccctatattctataaaaagataaaat Segments: 2.3.3.0 2582 2591 =CPE_bind= 1.1.2.0 2584 2593 ====CREB== 1.1.1.6 2585 2594 ==CRE-BP1= 4.5.1.0 2621 2630 ====TBP=== 4.3.1.1 2622 2631 ===RSRFC4= 2.2.1.1 2627 2636 ===GATA-1= 187
9. Appendix
1.1.3.0 2635 2644 =C/EB ======seq( 2640.. 2699) taattcttttatttttgtgtgaatttttattttgcattttcatttttaggtgcatcagtg Segments: 1.1.3.0 2635 2644 Pbeta 1.1.1.5 2644 2653 ====GCN4== 3.1.2.2 2644 2653 ===Oct-1== 3.3.2.0 2645 2654 ===HNF-3== 9.9.51 2658 2667 ====ATF=== 1.1.3.0 2663 2672 =C/EBPalp= 3.1.2.2 2667 2678 ====Oct-1A== 2.2.1.1 2668 2677 ===GATA-1= 9.9.539 2668 2677 ====NF-1== 3.5.3.0 2677 2686 ===ICSBP======seq( 2700.. 2759) cactgaaaaacctcacatcacaagattcagctaacatgaaaatagcagtgtgcacataca Segments: 3.1.2.2 2733 2742 ===Oct-1== 2.3.1.0 2745 2754 ====Sp1======seq( 2760.. 2819) gacatttaaagccaaggggcactgcacaaagaatttctgtgttcttgtgccttgggaata Segments: 9.9.539 2766 2777 =====NF-1=== 2.3.1.0 2770 2779 ====Sp1======seq( 2820.. 2879) aaaggaaaccccacgaaaaggcctccaagagctgatatgtgtgttgctccacctcgtcca Segments: 2.3.1.0 2824 2833 ====Sp1=== 2.3.1.0 2840 2849 ====Sp1=== 2.3.1.0 2865 2874 ====Sp1======seq( 2880.. 2939) ctcgtctctgctcattcgtgtgcctgtgctgggaaagaaagcacaaaggatgccgtgatg Segments: 1.1.3.0 2939 2948 ======seq( 2940.. 2999) ctgtgtaaatagtgaggccagcagtaaagaactgggttcttgttctgactttatcactcg Segments: 1.1.3.0 2939 2948 C/EBPalp= 2.3.1.0 2952 2961 ====Sp1=== 2.3.1.0 2967 2976 ====Sp1=== 2.1.1.1 2977 2986 =====GR=== 2.2.1.1 2990 2999 ===GATA-1= 1.1.1.2 2998 3007 == 9.9.32 2998 3007 == 1.1.1.1 2998 3008 ======seq( 3000.. 3059) ctgagtaacttcagattccctctggttctcatttaagccaagcgagcatttctaacacga Segments: 1.1.1.2 2998 3007 =c-Fos== 9.9.32 2998 3007 ==AP-1== 1.1.1.1 2998 3008 ==c-Jun== 9.9.29 3000 3009 ====AP-1== 1.1.1.5 3001 3010 ====AP-1== 9.9.590 3013 3022 =NF-kappaB 1.3.1.2 3024 3033 ====USF=== 4.6.1.0 3046 3055 ===SOX-9======seq( 3060.. 3119) gttctctgagatttgcttcagtaaattatggtacttcacaatttatattccaggtaagcc Segments: 3.1.2.2 3068 3079 ====Oct-1=== 1.1.3.0 3072 3081 =C/EBPalp= 1.1.3.0 3093 3102 =C/EBPalp= 2.3.1.0 3109 3118 ====Sp1=== 1.1.5.2 3117 3126 ======seq( 3120.. 3179) ccgtggcaaaggctcaacagcaatagtggagcccgagtaaaaacacgtctcctgatggtc Segments: 1.1.5.2 3117 3126 TAF-1== 188
9. Appendix
2.1.1.1 3171 3180 =====GR======seq( 3180.. 3239) cgatgggagggatcttgagggccaggaggttagcaagagcccatgtgtgccacttcttcc Segments: 2.1.1.1 3171 3180 = 2.3.1.0 3182 3193 =====Sp1==== 9.9.539 3197 3206 ====NF-1== 2.3.1.0 3197 3207 =====Sp1=== 1.1.3.0 3210 3219 =C/EBPalp= 1.3.1.2 3218 3227 ====USF=== 9.9.539 3222 3231 ====NF-1== 2.3.1.0 3228 3237 ====Sp1=== 2.3.1.0 3238 3247 ======seq( 3240.. 3299) cccagggaccctccctgacttctcctggaaagcccataggcatgtctctctcttacttcc Segments: 2.3.1.0 3238 3247 ==Sp1=== 2.3.1.0 3245 3258 ======Sp1===== 1.1.3.0 3255 3264 =C/EBPalp= 2.3.1.0 3256 3265 ====Sp1=== 4.1.1.0 3265 3274 =NF-kappa= 9.9.590 3265 3274 =NF-kappaB 3.5.2.0 3294 3303 ====PU 2.3.1.0 3295 3304 ====S ======seq( 3300.. 3359) tcttcaggactcattggcctcctttccctctccagactggcacttaagctgaactttatg Segments: 3.5.2.0 3294 3303 .1== 2.3.1.0 3295 3304 p1=== 9.9.539 3311 3320 ====NF-1== 2.3.1.0 3320 3333 ======Sp1===== 2.3.1.0 3331 3340 ====Sp1=== 9.9.539 3336 3345 ====NF-1== 2.1.2.1 3347 3356 =RAR-alph= 2.1.2.3 3347 3356 =REV-ErbA= 2.1.2.11 3347 3356 =HNF-4alp= 3.1.2.1 3356 3365 ===P 2.3.1.0 3359 3368 ======seq( 3360.. 3419) aatgggcaaagaattggtcaggaatgaaagttaaaacaaaacagagaccaacaaacaaaa Segments: 3.1.2.1 3356 3365 it-1a= 2.3.1.0 3359 3368 ===Sp1=== 2.1.1.1 3363 3372 =====GR=== 9.9.1202 3372 3381 ====CP1=== 3.6.1.0 3379 3388 ====TEC1== 3.3.2.0 3392 3401 ===HNF-3== 1.1.3.0 3393 3402 =C/EBPalp= 1.1.3.0 3409 3418 =C/EBPalp= 9.9.590 3418 3427 =N ======seq( 3420.. 3479) atcccaaacactgaggtgaattttaacctctaaaatgcccactgtaacaatagtgaaggt Segments: 9.9.590 3418 3427 F-kappaB 3.5.1.2 3422 3431 ====RAP1== 4.3.1.1 3438 3447 ===MEB-1== 4.3.1.2 3438 3447 ====GLO======seq( 3480.. 3539) tctgcaggcctcctggtcaatgtaaacccacttttcctctaaagcacaatttgatcatta Segments: 2.3.1.0 3484 3493 ====Sp1=== 2.1.1.4 3493 3502 =====ER=== 3.1.2.2 3496 3505 ===Oct-1== 4.1.1.0 3510 3519 =NF-kappa= 3.1.1.12 3532 3541 ===HNF-1 1.1.3.0 3534 3547 ===C/E
189
9. Appendix
seq( 3540.. 3599) tttattaatgaatcacacttctcctgtggacagaatccttcctgtgttctcttctatcag Segments: 3.1.1.12 3532 3541 == 1.1.3.0 3534 3547 BPalpha= 1.1.1.1 3545 3554 ===c-Jun== 1.1.1.5 3546 3555 ====GCN4== 3.5.2.0 3576 3585 =c-Ets-1_= 2.1.1.1 3580 3589 =====GR=== 2.1.1.2 3582 3591 =====PR=== 2.2.1.1 3590 3601 ====GATA-1 ======seq( 3600.. 3659) aaatgcatcccattcattcacctatgagctgacttttctgcaacctccagtgtgaaaaca Segments: 2.2.1.1 3590 3601 == 9.9.539 3603 3612 ====NF-1== 3.1.2.1 3611 3620 ===Pit-1a= 1.3.1.2 3623 3632 ====USF=== 3.5.2.0 3635 3644 ===Erg-1======seq( 3660.. 3719) ccttcctcttgtttcaagaagagttatcaattagtacaggccccaaacaggggtggaaat Segments: 2.3.1.0 3660 3669 ====Sp1=== 3.5.2.0 3660 3669 ====PU.1== 9.9.322 3661 3670 ====GCR1== 2.1.1.2 3675 3684 =====PR=== 2.3.1.0 3694 3703 ====Sp1=== 2.3.1.0 3707 3716 ====Sp1=== 3.1.2.2 3716 3730 ======seq( 3720.. 3779) ctgcattgcattacttgtgacagagaaaatggcagacataaccagaaacagggaacagga Segments: 3.1.2.2 3716 3730 ==Oct-1==== 9.9.539 3720 3729 ====NF-1== 2.1.1.4 3732 3741 =====ER=== 3.1.1.2 3741 3750 ====Ftz=== 3.5.2.0 3772 3783 ==c-Ets- 1.1.3.0 3773 3782 =C/EBPa 9.9.590 3773 3782 =NF-kap ======seq( 3780.. 3839) aattttggataaacagtagtggtggttatgaagcagctgaagcgaatcccagacactaga Segments: 3.5.2.0 3772 3783 1_68 1.1.3.0 3773 3782 lp= 9.9.590 3773 3782 paB 9.9.539 3781 3790 ====NF-1== 1.1.3.0 3836 3845 =C/E ======seq( 3840.. 3899) gtttgtaacagcatcttgaattgcaggattaaaggtaactacttcccacttagacataat Segments: 1.1.3.0 3836 3845 BPalp= 3.1.1.2 3854 3863 ====Ftz======seq( 3900.. 3959) ctccattcaactcaaaatttgagagtagatttatagacatggcattgtgttctataatag Segments: 1.1.5.3 3935 3944 ====GBF1== 2.1.1.1 3943 3952 =====GR=== 9.9.539 3955 3964 ====N ======seq( 3960.. 4019) tgccagaaagcaataactaaagacacttgtggtgcacatttgaatgtggagaaaagaaca Segments: 9.9.539 3955 3964 F-1== 3.1.1.12 3971 3980 ===HNF-1== 3.1.2.2 3997 4006 ===Oct-1== 4.3.2.0 4002 4011 ====SRF=== 1.1.3.0 4004 4013 =C/EBPalp= 2.1.1.1 4013 4022 =====GR ======seq( 4020.. 4079) gaacacataataaataacttgagtggtttgctctgcataccatcatggtttcacagtaca 190
9. Appendix
Segments: 2.1.1.1 4013 4022 === 3.1.1.12 4028 4037 ===HNF-1C= 9.9.15 4028 4037 ====AFP1== 1.1.3.0 4029 4038 =C/EBPalp= 1.1.3.0 4043 4052 =C/EBPalp= 3.1.2.2 4048 4057 ===Oct-1== 2.3.1.0 4078 4087 ======seq( 4080.. 4139) cggaggtgaaatgaatgattctcatttacagactcaataactgattttttttcaggctat Segments: 2.3.1.0 4078 4087 ==Sp1=== 3.1.1.12 4113 4122 ===HNF-1== 1.1.3.0 4114 4123 =C/EBPdel= 1.1.3.0 4123 4132 =C/EBPalp= 2.3.2.2 4123 4132 =====Hb======seq( 4140.. 4199) caaacagcttaagttttttcaacacaagagaacaatgcctctctcattgggtttgcggtg Segments: 9.9.173 4180 4189 ====CTF=== 9.9.233 4180 4189 ====EFI======seq( 4200.. 4259) tagactccgaacagagtgtttaatatacacagctcaccctgctcttcttgggggaacagt Segments: 2.1.1.1 4208 4217 =====GR=== 4.5.1.0 4220 4229 ====TBP=== 2.3.1.0 4229 4238 ====Sp1=== 2.3.1.0 4236 4245 ====Sp1======seq( 4260.. 4319) cctgattaactttagattctaagagaaccataggaggcctgagattcacttgaaactgag Segments: 2.3.1.0 4292 4301 ====Sp1=== 3.5.3.0 4311 4320 ===ICSBP======seq( 4320.. 4379) gcttttaagagactttgtttatggccttcctcgttagaatagctcttttcttgtgaagga Segments: 3.5.3.0 4311 4320 = 2.1.1.1 4362 4371 =====GR=== 2.2.1.1 4364 4373 ===GATA-1======seq( 4380.. 4439) aaacactaagctggtttcaaaaaaacgtactggccttgtcttcagctccataattcctat Segments: 1.1.3.0 4395 4404 =C/EBPalp= 2.3.2.2 4397 4406 ===LyF-1== 2.2.1.1 4411 4420 ===GATA-1= 1.1.3.0 4437 4446 =C/ ======seq( 4440.. 4499) gtatgctcttgggagggctaagtggataaaaactcagacctggttgtctctgtcccctag Segments: 1.1.3.0 4437 4446 EBPalp= 2.3.1.0 4447 4456 ====Sp1======seq( 4500.. 4559) ttcttcattctcctccaggcagaaagatggcaggccatagtaacgacagatttcagatac Segments: 3.6.1.0 4501 4510 ====TEC1== 2.3.1.0 4505 4514 ====Sp1=== 2.3.1.0 4523 4532 ====YY1======seq( 4560.. 4619) tgagatgtttcctgagaggacaacctgacagaagagttaggtgggggtgctgaggcacca Segments: 1.1.3.0 4565 4574 ===C/EBP== 2.3.1.0 4599 4608 ====Sp1=== 3.5.1.2 4599 4608 ====RAP1== 9.9.77 4599 4608 =CACCC-bi= 1.1.3.0 4605 4616 ==C/EBPgamma 9.9.29 4607 4616 ====AP-1== 9.9.31 4607 4616 ====AP-1== 1.1.3.0 4616 4625 =C/E 191
9. Appendix
3.5.2.0 4617 4626 =c- 1.2.2.0 4618 4627 ======seq( 4620.. 4679) ggaaatgtccagatccacccctttaatggtaactaccttcttggaagctctgcactcccc Segments: 1.1.3.0 4616 4625 BPalp= 3.5.2.0 4617 4626 Ets-1_= 1.2.2.0 4618 4627 ==MyoD== 4.3.2.0 4624 4633 ====SRF=== 2.3.1.0 4631 4643 ======Sp1==== 4.3.2.0 4639 4648 ====SRF=== 3.1.1.2 4640 4649 ===Zen-1== 2.3.1.0 4665 4674 ====Sp1=== 2.3.1.0 4672 4686 ======S 3.5.2.0 4678 4687 ======seq( 4680.. 4739) acctcttcttctttcccctcccactctctgtctcttttctggtccgtatctggcctatca Segments: 2.3.1.0 4672 4686 p1===== 3.5.2.0 4678 4687 =Elk-1== 2.3.1.0 4693 4703 =====Sp1=== 2.3.1.0 4729 4738 ====Sp1=== 2.2.1.1 4733 4742 ===GATA ======seq( 4740.. 4799) tagtaagaggtcagtctgttttccacacttggatagtctcattcaaaaatttttgtcaat Segments: 2.2.1.1 4733 4742 -1= 2.1.2.1 4743 4752 =RAR-alph= 2.1.2.2 4743 4752 =RXR-beta2 2.1.1.4 4747 4756 =====ER=== 4.3.2.0 4762 4771 ====SRF=== 9.9.537 4766 4775 ====NF-1== 1.1.3.0 4781 4790 =C/EBPalp= 2.3.2.2 4786 4795 =====Hb=== 3.1.1.2 4787 4796 ===HOXA4== 1.1.3.0 4792 4801 =C/EBPal ======seq( 4800.. 4859) ggacaagtcataagaaacccttccattttcgggctcgttgacgtcaccaaggaggcgata Segments: 1.1.3.0 4792 4801 p= 3.5.1.2 4816 4825 ====REB1== 1.1.3.0 4820 4829 =C/EBPalp= 3.5.1.2 4835 4844 ===Adf-1== 9.9.51 4835 4844 ====ATF=== 2.1.2.3 4836 4845 =REV-ErbA= 1.1.2.0 4836 4846 ====CREB=== 1.1.1.6 4837 4846 ==CRE-BP1= 1.1.1.1 4838 4847 ===c-Jun== 2.3.3.0 4838 4847 =CPE_bind= 2.3.1.0 4847 4856 ====Sp1=== 1.1.3.0 4857 4866 =C/ 2.2.1.1 4859 4868 = 3.1.2.2 4859 4868 ======seq( 4860.. 4919) aatatctgttgatataattggatgtgagattcagtgttgaaatagcagaactctgtccct Segments: 1.1.3.0 4857 4866 EBPalp= 2.2.1.1 4859 4868 ==GATA-1= 3.1.2.2 4859 4868 ==Oct-1======seq( 4920.. 4979) cgctccttggcagggccctattatttatgcaggagcagaggcagcacgcaatcgagctgt Segments: 9.9.173 4921 4930 ====CTF=== 9.9.539 4925 4934 ====NF-1== 2.3.1.0 4927 4937 =====Sp1=== 4.5.1.0 4941 4950 ====TBP=== 1.1.3.0 4944 4953 =C/EBPalp======192
9. Appendix seq( 4980.. 5039) caagagagcgtcagcttattaggcaaatgctgcgtgctttctgaagagggtcgacattat Segments: 1.1.3.0 4996 5005 =C/EBPalp= 2.3.1.0 5021 5031 =====Sp1=== 1.1.3.0 5035 5049 ===C/ ======seq( 5040.. 5099) aaaatctcactccaggctctggtgtggagaaactcagagcccaagtacgttgagagactg Segments: 1.1.3.0 5035 5049 EBPalpha== 1.1.3.0 5061 5070 =C/EBPalp= 4.1.1.0 5066 5075 =====Dl======seq( 5100.. 5159) aagagaaagggaaaaggcaaaagaaaaaaagaagagaaaggagaagaggaagaaaacctg Segments: 3.5.3.0 5104 5113 ===IRF-1== 9.9.428 5104 5113 ===ISGF-3= 9.9.701 5107 5116 =PTF1-beta 1.1.3.0 5109 5118 =C/EBPalp= 2.3.2.2 5119 5128 =====Hb=== 3.1.2.2 5123 5132 ===Oct-1== 2.2.1.1 5129 5138 ===GATA-1= 3.5.3.0 5129 5138 ===ICSBP== 2.3.1.0 5140 5151 =====Sp1==== 3.5.2.0 5143 5152 ===Elf-1== 2.3.1.0 5156 5165 ======seq( 5160.. 5219) caggaggcatcctgagagaagtccctctgcagaggcagcagtgcgggctcacctaccaag Segments: 2.3.1.0 5156 5165 Sp1=== 2.3.1.0 5162 5171 ====Sp1=== 4.1.1.0 5175 5184 =NF-kappa= 9.9.590 5175 5184 =NF-kappaB 2.3.1.0 5180 5189 ====Sp1=== 2.3.1.0 5217 5227 ======seq( 5220.. 5279) ggaggagaaggtaggcagcgcctagacgggcgccaccaactttgtgctgcctgagctgct Segments: 2.3.1.0 5217 5227 ==Sp1=== 2.3.1.0 5233 5242 ====Sp1=== 2.3.1.0 5244 5257 ======Sp1===== 1.2.1.0 5265 5274 =====E4=== 2.1.1.4 5269 5278 =====ER======seq( 5280.. 5339) gtggtgagccccggagccagctgcccatgtgctggaatgcctgtgcctatgcatgtatgt Segments: 2.3.1.0 5288 5298 =====Sp1=== 1.3.1.2 5293 5302 ====USF=== 1.2.1.0 5295 5304 =====E1=== 1.2.2.0 5295 5304 ===Myf-3== 2.3.1.0 5295 5304 ====Sp1=== 1.3.1.2 5301 5310 ====USF=== 3.5.1.2 5333 5342 ====RAP ======seq( 5340.. 5399) gtgtcgctaactgtgccttaaaattccgatgacagtggcgatttgaaaaagcgaagttag Segments: 3.5.1.2 5333 5342 1== 1.1.3.0 5379 5388 =C/EBPalp======seq( 5400.. 5459) acggcggctgctcatctttatccactcaatccaatctgccactcactgctcatagtctgt Segments: 3.5.1.2 5401 5410 ===Adf-1== 2.2.1.1 5407 5416 ===GATA-1= 2.1.1.1 5433 5442 =====GR=== 2.3.1.0 5438 5447 ====Sp1=== 2.2.1.1 5455 5464 ===GA ======seq( 5460.. 5519) gcaaagaatggctcccctattgcatcccatgtccccaagcaaacggagtaagggcaggaa Segments: 193
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2.2.1.1 5455 5464 TA-1= 1.6.1.0 5467 5476 ====AP-2== 1.3.1.2 5485 5494 ====USF=== 3.6.1.0 5515 5524 ====T ======seq( 5520.. 5579) tggagacagagaaggttgttctcaatttggcagaaaaggatgtccgaaagggggcgatta Segments: 3.6.1.0 5515 5524 EC1== 1.1.3.0 5538 5547 =C/EBPalp= 9.9.535 5542 5551 ====NF-1== 9.9.539 5543 5552 ====NF-1== 2.1.1.1 5545 5554 =====GR=== 3.5.2.0 5553 5562 ====PEA3== 2.3.1.0 5566 5575 ====Sp1=== 3.5.1.2 5577 5586 ======seq( 5580.. 5639) gggtgtgcgacagcttaaacctgtggcacttgtccgggctcagggaagtcggtttaggga Segments: 3.5.1.2 5577 5586 =RAP1== 1.1.3.0 5584 5593 =C/EBPbeta 9.9.539 5603 5612 ====NF-1== 2.3.1.0 5614 5623 ====Sp1=== 1.1.1.6 5636 5645 ==CR 1.1.2.0 5636 5645 ======seq( 5640.. 5699) agacgtttgggaggtccttaggaagaggagccaggggttgtcccttctaggtctccaaag Segments: 1.1.1.6 5636 5645 E-BP1= 1.1.2.0 5636 5645 CREB== 2.3.1.0 5648 5657 ====Sp1=== 2.3.1.0 5661 5673 ======Sp1==== 2.3.1.0 5672 5681 ====Sp1=== 2.3.1.0 5679 5688 ====Sp1=== 4.3.2.0 5694 5703 ====SR ======seq( 5700.. 5759) aagggtcaccgcgggctcgcaccagttgagctttgcaggtacctagcttcagcaccgcgg Segments: 4.3.2.0 5694 5703 F=== 9.9.32 5705 5714 ====AP-1== 2.3.1.0 5709 5718 ====Sp1======seq( 5760.. 5819) acagcgtcaccgaagcctagagcctgtcttgtctgtgggtgtccgataggaagccccgct Segments: 2.3.3.0 5762 5771 =CPE_bind= 4.4.1.0 5767 5776 =====E2=== 3.5.1.2 5790 5799 ====RAP1== 2.3.1.0 5791 5800 ====Sp1=== 2.2.1.1 5802 5811 ===GATA-1= 2.3.1.0 5809 5822 ======Sp1======seq( 5820.. 5879) gcaccttccagctgagctaaactctgaccaatcttacctttctcccccaccttctctctc Segments: 2.3.1.0 5809 5822 === 2.1.1.4 5840 5849 =====ER=== 2.3.1.0 5861 5873 ======Sp1==== 2.3.1.0 5874 5886 ======2.3.3.0 5876 5885 =CPE ======seq( 5880.. 5906) ccccgacctcaacctcggtgcttcag Segments: 2.3.1.0 5874 5886 Sp1==== 2.3.3.0 5876 5885 _bind= 2.3.1.0 5880 5889 ====Sp1=== 2.3.1.0 5886 5895 ====Sp1=== 3.1.2.2 5898 5906
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9.7. Northern Blot
Murine CRH Promoter Region and Appropriate Primers Used for Amplification of the CRH Probe
Sequence Origin: ENSEMBL Database Chromosome 3:19,593,401 - 19,595,396, Genomic DNA
Illustrated CRH Promoter Region: 5,000 bp (Start of CRH-Exon 1) - 6,996 bp (End of CRH-Exon 2)
Cloned CRH Probe (357 bp): 6,036 bp – 6,392 bp (Pr.9 – Pr.10) Amplification from corresponding mRNA by RT-PCR
Legend: CRH-Exon 1/2 Primer CRH-Ex2-Forward, Pr. 9 Primer CRH-Ex2-Reverse, Pr. 10 Probe (357 bp)
AGGCAAATGCTGCGTGCTTTCTGAAGAGGGTCGACATTATAAAATCTCACTCCAGGCTCTGGTG TGGAGAAACTCAGAGCCCAAGTACGTTGAGAGACTGAAGAGAAAGGGAAAAGGCAAAAGAAAAA AAGAAGAGAAAGGAGAAGAGGAAGAAAACCTGCAGGAGGCATCCTGAGAGAAGTCCCTCTGCAG AGGCAGCAGTGCGGGCTCACCTACCAAGGGAGGAGAAGGTAGGCAGCGCCTAGACGGGCGCCAC CAACTTTGTGCTGCCTGAGCTGCTGTGGTGAGCCCCGGAGCCAGCTGCCCATGTGCTGGAATGC CTGTGCCTATGCATGTATGTGTGTCGCTAACTGTGCCTTAAAATTCCGATGACAGTGGCGATTT GAAAAAGCGAAGTTAGACGGCGGCTGCTCATCTTTATCCACTCAATCCAATCTGCCACTCACTG CTCATAGTCTGTGCAAAGAATGGCTCCCCTATTGCATCCCATGTCCCCAAGCAAACGGAGTAAG GGCAGGAATGGAGACAGAGAAGGTTGTTCTCAATTTGGCAGAAAAGGATGTCCGAAAGGGGGCG ATTAGGGTGTGCGACAGCTTAAACCTGTGGCACTTGTCCGGGCTCAGGGAAGTCGGTTTAGGGA AGACGTTTGGGAGGTCCTTAGGAAGAGGAGCCAGGGGTTGTCCCTTCTAGGTCTCCAAAGAAGG GTCACCGCGGGCTCGCACCAGTTGAGCTTTGCAGGTACCTAGCTTCAGCACCGCGGACAGCGTC ACCGAAGCCTAGAGCCTGTCTTGTCTGTGGGTGTCCGATAGGAAGCCCCGCTGCACCTTCCAGC TGAGCTAAACTCTGACCAATCTTACCTTTCTCCCCCACCTTCTCTCTCCCCCGACCTCAACCTC GGTGCTTCAGAGAGCGCCCCTAACATGCGGCTGCGGCTGCTGGTGTCCGCGGGCATGCTGCTGG TGGCTCTGTCGTCCTGCCTGCCTTGCAGGGCCCTGCTCAGCAGGGGATCCGTCCCCCGAGCGCC GCGGGCCCCGCAGCCCTTGAATTTCTTGCAGCCGGAGCAGCCCCAGCAACCTCAGCCGGTTCTG ATCCGCATGGGTGAAGAATACTTCCTCCGCCTGGGGAATCTCAACAGAAGTCCCGCTGCTCGGC TGTCCCCCAACTCCACGCCCCTCACCGCGGGTCGCGGCAGCCGCCCCTCGCACGACCAGGCTGC 195
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GGCTAACTTTTTCCGCGTGTTGCTGCAGCAGCTGCAGATGCCTCAGCGCTCGCTCGACAGCCGC GCGGAGCCGGCCGAACGCGGCGCCGAGGATGCCCTCGGTGGCCACCAGGGGGCGCTGGAGAGGG AGAGGCGGTCGGAGGAGCCGCCCATCTCTCTGGATCTCACCTTCCACC
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9.8. Abbreviations
ACE Angiotensin converting enzyme ACTH Adrenocorticotropic hormone ADP Adenosine diphosphate AGM Aorta-gonad-mesonephros Ang Angiotensin ANP Atrial natriuretic peptide AP-1 Activator Protein 1 APS Ammonium persulfate AQP Aquaporin AS Aldosterone synthase AT Angiotensin receptor ATF1 cAMP-dependent transcription factor ATP Adenosine triphosphate AVP Arginine vasopressin B Basal
BRCA1 Breast Cancer 1 BSA Bovine serum albumine BW Body weight Ca2+ Calcium CaMK Ca2+/calmodulin-dependent protein kinase cAMP Cyclic adenosine monophosphate Cat. No. Catalog number Cbl Casitas B-lineage lymphoma CD Cluster of differentiation CK Creatine Kinase Cl- Chloride CLIP Corticotropin-like intermediate peptide CNS Central nervous system Cre Causes recombination CRE cAMP-responsive element CREB cAMP-responsive element binding protein
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CRH Corticotropin-releasing hormone
CRHProm CRH promoter reporter including RE-1/NRSE regulatory element
CRHProm-RE CRH promoter reporter lacking RE-1/NRSE regulatory element CRH-R Corticotropin-releasing hormone receptor DAPI 4',6-Diamidino-2-phenylindole DEPC Diethylpyrocarbonate DHEA Dehydroepiandrosterone DMEM Dulbecco’s modified Eagle's medium DNA Deoxyribonucleic acid dUTP Deoxyuridine triphosphate ECG Electrocardiography ECL Enhanced chemoluminescence EDTA Ethylenediaminetetraacetic acid EGF Epidermal growth factor EGFR Epidermal growth factor receptor EGTA Ethylene glycol tetraacetic acid EIA Enzyme immuoassay ELISA Enzyme-linked immunosorbent assay En2 intron 1 Engrailed2 intron 1 Ena Enabled ENaC Epithelial sodium channel ENU N-Ethyl-N-nitrosourea EPSC Excitatory postsynaptic current EPSP Excitatory postsynaptic potential ERK Extracellular signal-regulated kinase ES cell Embryonic stem cell EST Expressed sequence tag Ets E-twenty six Eve EVH1 enhanced EVH1 Ena/VASP homology 1 domain Ex Exon FBS Fetal bovine serum FGF Fibroblast growth factor FGFR Fibroblast growth factor receptor
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FGFRL Fibroblast growth factor receptor-like FLP Flippase FRS2 Fibroblast growth factor receptor substrate 2 FRT Flippase recognition target FSH Follicle-stimulating hormone GABA γ-Aminobutyric acid GAB1 GRB2-associated binding protein 1 GDP Guanosine diphosphate GF Growth factor GFR Glomerular filtration rate GGT γ-Glutamyltransferase GH Growth hormone GHRH Growth hormone-releasing hormone GnRH Gonadotropin-releasing hormone GOT/AST Glutamate oxaloacetate transaminase/Aspartate aminotransferase
GPT/ALT Glutamate pyruvate transaminase/Alanine aminotransferase GR Glucocorticoid receptor GRB2 Growth factor receptor-bound protein 2 GRE Glucocorticoid response element GTP Guanosine triphosphate h Hour H&E Hematoxylin & Eosin HCC Hepatocellular carcinoma HEK Human embryonic kidney HEPES 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid HET Heterozygous HGF Hepatocyte growth factor HPA Hypothalamic-pituitary-adrenal HSD11B2/11β-HSD type II 11β-Hydroxysteroid-dehydrogenase type 2 HSD3B/ 3β-HSD 3β-Hydroxysteroid-dehydrogenase IGF-1 Insulin-like growth factor 1 IgG Immunoglobulin G IGTC International gene trap consortium IMPC International mouse phenotyping consortium
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IL Interleukin IRS-1/2 Insulin receptor substrate 1/2 JNK c-Jun N-terminal kinase K+ Potassium KBD c-Kit binding domain kDa Kilodalton KO Knockout lacZ β-Galactosidase gene LATS Large tumor suppressor LH Luteinizing hormone LoxP Locus of X-over P1 LPH Lipotropin Luc Luciferase M Marker MAPK Mitogen-activated protein kinase MAPKK/MEK Mitogen-activated protein kinase kinase MAPKKK Mitogen-activated protein kinase kinase kinase MARKK Microtubule affinity-regulating kinase-activating kinase MCR Melanocortin receptor mGluR Metabotropic glutamate receptor mHypoE-44 Embryonic mouse hypothalamic cell line N44 min Minute MMP Matrix metalloproteinases MNK MAPK interacting kinases MOPS 3-(N-Morpholino)propanesulfonic acid MSH Melanocyte-stimulating hormone MSK Mitogen- and stress-activated kinase n Number Na+ Sodium NBR1 Neighbor of BRCA1 neoR Neomycin resistance gene NF1 Neurofibromin 1 NF-КB Nuclear factor `kappa-light-chain-enhancer´ of activated B cells
NGF Nerve growth factor
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NGF1B Nerve growth factor 1 B NGFR Nerve growth factor receptor NPC Neural progenitor cell NURR1 Nuclear receptor related 1 OCD Obsessive-compulsive disorder p Probability P450aldo/CYP11B2 Aldosterone synthase P450c11/CYP11B1 11β-Hydroxylase P450c17/CYP17A1 17α-Hydroxylase P450c21/CYP21A2 21α-Hydroxylase P450scc/CYP11A1 Cholesterol side-chain cleavage monooxygenase pA Simian virus 40 polyadenylation site PBS Phosphate buffered saline PCR Polymerase chain reaction PDGF Platelet-derived growth factor receptor PDGFR Platelet-derived growth factor PDV Tumorigenic murine keratinocyte cell line PFA Paraformaldehyde PKA Protein kinase A PKC Protein kinase C 3- PO4 Phosphate POMC Proopiomelanocortin Pr. Primer PRL Prolactin PSD 95 Postsynaptic density protein 95 PTB Phosphotyrosine-binding pUC pUC vector backbone PVN Paraventricular nucleus Rab Ras-related protein Raf Rapidly accelerated fibrosarcoma Ras Rat sarcoma RAS Renin-Angiotensin system RasGAP Ras GTPase-activating protein RasGEF Ras guanine nucleotide exchange factor RBM Raf-binding motif
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RE-1/NRSE Repressor element-1/neuron-restrictive silencing element RhoA Ras homolog gene family, member A RNA Ribonucleic acid RNAi RNA interference ROCK Rho-associated protein kinase ROMK Renal outer medullary potassium channel RSK Ribosomal S6 kinase RTK Receptor tyrosine kinase RT-PCR Reverse transcription polymerase chain reaction s Second SA Splice acceptor SAPAP3 SAP90/PSD95-associated protein 3 SCF Stem cell factor SCN Suprachiasmatic nucleus SD Standard deviation SDS Sodium dodecyl sulphate SDS-PAGE Sodium dodecyl sulphate-polyacrylamide gel electrophoresis SGK1 Serum and glucocorticoid-inducible kinase 1 SH2 Src homology 2 SH3 Src homology 3 SHC Src homology and collagen protein SHP2/PTPN11 Src homology 2 domain containing protein-tyrosine phosphatase 2 / Tyrosine-protein phosphatase non-receptor type 11 siRNA Small interfering RNA SLITRK5 SLIT and NTRK-like protein-5 SON Supraoptic nucleus SOS Son of sevenless SPR2 SPRED2 SPRED Sprouty-related protein with EVH1 domain Src Sarcoma SSC Saline-sodium citrate StAR Steroidogenic acute regulatory protein STAT Signal transducers and activators of transcription TASK TWIK-related acid-sensitive K+ channel
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TEMED Tetramethylethylendiamin TESK Testis-specific protein kinase 1 TGF-β1 Transforming growth factor β 1 TNF α Tumor necrosis factor α TRH Thyrotropin-releasing hormone TSH Thyroid-stimulating hormone TUNEL Terminal deoxynucleotidyl transferase dUTP nick end labeling U Unit uPA Urokinase type plasminogen activator UV Ultraviolett V1a/1b/2R Vasopressin receptor 1a, 1b, 2 VASP Vasodilator-stimulated phosphoprotein VEGF Vascular endothelial growth factor VEGFR Vascular endothelial growth factor receptor WASP Wiskott-Aldrich syndrome protein WD Water deprivation WT Wild-type X-Gal 5-Bromo-4-chloro-3-indolyl-beta-D-galactopyranoside β-gal β-galactosidase β-geo Fusion gene composed of β-galactosidase gene (lacZ) and neomycin resistance gene (neoR)
203